Antifungal sordaridin derivatives

ABSTRACT

Compounds of the formula (I) ##STR1## and pharmaceutically acceptable salts or metabolically labile derivatives thereof, processes for their preparation, their use as antifungal agents and intermediates for use in their preparation.

This invention relates to novel sordarin derivatives having antifungalactivity, to processes for their preparation, to pharmaceuticalcompositions containing them and to their use in medicine, moreparticularly in the prevention or treatment of diseases in animals,including humans, caused by fungal infection.

British Patent Specification No. 1,162,027 describes the preparation ofan antibiotic, SL2266, by the cultivation of the strain NRRL 3196 of thefungus species Sordaria araneosa. SL 2266, later named sordarin, isreported to have fungistatic activity. The same research group alsodescribed in Helvetica Chimica Acta (1971), 51, 119-120 the degradationof sordarin to sordaricin. Published Japanese Patent Application No. J62040292A describes the preparation of an antibiotic, zofimarin, which isreported to have antifungal activity.

Sordarin, sordaricin and zofimarin may be represented by formula (A)below ##STR2## where OR as ##STR3## describes sordarin; OR as OHdescribes sordaricin; and

OR as ##STR4## describes zofimarin.

Although sordarin and zofimarin exhibit antifungal activity, bothcompounds are only moderately active and have limited spectra of actionwhen tested against a battery of fungal organisms. We now describehereinafter a novel group of fungicidal sordarin derivatives whichexhibit excellent antifungal activity and a broad spectrum of action.Thus, according to a first aspect of the present invention, we providecompounds of formula (I) ##STR5## and pharmaceutically acceptable saltsand solvates (e.g. hydrates) or metabolically labile derivativesthereof,

wherein R¹ represents hydrogen, halogen, hydroxyl or C₁₋₄ alkoxy;

R² represents hydrogen, halogen, hydroxyl, C₁₋₁₀ alkoxy, C₁₋₁₀alkylthio, C₁₋₆ alkoxyC₁₋₄ alkoxy, arylC₁₋₆ alkyloxy, arylC₃₋₆alkenyloxy, azido, NR⁵ COR⁵ (where each R⁵ is independently hydrogen orC₁₋₆ alkyl), OR⁶ (where R⁶ is a cyclic ether containing 4 to 8 atomslinked to the oxygen atom via a ring carbon atom adjacent to the ringoxygen atom) or a group ##STR6## where Y is oxygen, sulphur or NH, X iseither a bond, an oxygen atom or a moiety NR⁸ in which R⁸ is hydrogen orC₁₋₆ alkyl, and R⁷ is C₁₋₁₀ alkyl optionally containing one or twodouble bonds, aryl, arylC₁₋₄ alkyl, arylC₂₋₄ alkenyl, haloC₁₋₆ alkyl orC₁₋₆ alkoxyC₁₋₄ alkyl), and R³ represents hydrogen, or R² and R³ maytogether with the carbon atom to which they are attached represent C═Oor C═NOR⁹ (where R⁹ is C₁₋₆ alkyl); and

R⁴ represents hydroxyl, C₁₋₆ alkoxy or ##STR7## (where R⁷ is as definedabove); with the proviso that when R¹ represents a hydroxyl group in theaxial configuration and R⁴ is methoxy then R² cannot represent a groupin the axial configuration selected from hydroxyl and OCOCH═^(Z)CH--CH═^(E) CHCH₃.

Suitable pharmaceutically acceptable salts of the compounds of formula(I) include inorganic base salts such as alkali metal salts (for examplesodium and potassium salts) and ammonium salts and organic base salts.Suitable organic base salts include amine salts such as trialkylamine(e.g. triethylamine), dialkylamine (e.g. dicyclohexylamine), optionallysubstituted benzylamine (e.g. phenylbenzylamine or p-bromobenzylamine),procaine, ethanolamine, diethanolamine, N-methylglucosamine andtri(hydroxymethyl)methylamine salts and amino acid salts (e.g. lysineand arginine salts).

References hereinafter to a compound of formula (I) includes thatcompound and its pharmaceutically acceptable salts.

Other salts which are not pharmaceutically acceptable may be useful inthe preparation of compounds of formula (I) and these form a furtheraspect of the invention.

Metabolically labile derivatives of compounds of formula (I) arecompounds which are converted in the body into compounds of formula (1).Examples of such derivatives include conventional metabolically labileesters formed from the free carboxylic acid in the molecule.

It is to be understood that the present invention encompasses anyindividual isomers, including optical isomers, of compounds representedby formula (I) above as well as mixtures thereof, including wholly orpartially racemic mixtures thereof.

As used herein, the term "alkyl" as a group or part of a group means astraight or branched chain alkyl moiety comprising, for example, 1 to 8carbon atoms. Suitable examples include methyl, ethyl, n-propyl,i-propyl, n-butyl, s-butyl, t-butyl, n-hexyl and n-octyl.

As used herein, the term "aryl" as a group or part of a group meansphenyl or heteroaryl each optionally substituted by one or more (e.g. 1,2 or 3) atoms or groups selected from halogen, hydroxyl, C₁₋₆ alkyl,C₁₋₆ alkoxy or C₁₋₄ alkoxycarbonyl. The heteroaryl group may be a 5- or6-membered heteroaromatic ring containing one or more heteroatomsselected from nitrogen, oxygen and sulphur. Suitable examples ofheteroaryl groups include pyridyl, furyl, thienyl and pyrrolyl.

The term "halogen" or "halo" means herein fluorine, chlorine, bromine oriodine.

A haloC₁₋₆ alkyl group within R⁷ means a C₁₋₆ alkyl group containing oneor more (e.g. 1, 2 or 3) halogen atoms.

When R¹ represents a C₁₋₄ alkoxy group it may be, for example, a methoxygroup. R¹ may particularly represent hydrogen, fluorine, hydroxyl ormethoxy.

When R¹ is a halogen atom or a hydroxyl or C₁₋₄ alkoxy group, the R¹moiety is preferably sited in the axial configuration. Particularlypreferred are compounds of formula (I) in which R¹ is hydrogen orhydroxyl (especially when sited in the axial configuration).

Examples of the group R² include halogen (e.g. fluorine), C₁₋₁₀ alkoxy(for example a C₁₋₆ alkoxy group such as methoxy, ethoxy, n-propoxy orn-hexyloxy), C₁₋₆ alkylthio (for example a C₁₋₄ alkylthio group such asmethylthio), C₁₋₄ alkoxyC₁₋₄ alkoxy (for example a C₁₋₄ alkoxymethoxygroup such as methoxymethoxy), arylC₁₋₄ alkyloxy (for example aphenylC₁₋₄ alkyloxy group such as benzyloxy), phenylC₃₋₆ alkenyloxy (forexample OCH₂ CH═^(E) CHPh), azido, NR⁵ COR⁵ (where each R⁵ isindependently hydrogen or a C₁₋₄ alkyl group such as methyl), OR⁶ (whereR⁶ is, for example, a 2-tetrahydropyranyl group), OCOR⁷ where R⁷ isC₁₋₁₀ alkyl (e.g. methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl,t-butyl, 1-methylpentyl or n-heptyl), C₂₋₁₀ alkyl containing one or twodouble bond (for example a C₃ alkenyl group such as --C(CH₃)═CH₂, a C₆alkenyl group such as --C(CH₃)═C(CH₂)₂ CH₃ or a C₅ diene such as--CH═^(Z) CH--CH═^(E) CHCH₃), aryl (e.g. phenyl or p-tolyl), arylC₁₋₄alkyl (for example an arylmethyl group such as benzyl), arylC₂₋₄ alkenyl(for example an arylethenyl group such as PhCH═CH--), monohaloC₁₋₄ alkyl(for example a halopropyl group such as 3-chloropropyl), C₁₋₆alkoxymethoxy (e.g. methoxymethoxy or n-butoxymethoxy), OCO₂ R⁷ where R⁷is C₁₋₁₀ alkyl (e.g. n-octyl), ##STR8## where R⁷ is C₁₋₁₀ alkyl (forexample methyl or n-octyl) or arylC₁₋₄ alkyl (for example an arylmethylgroup such as benzyl) and R⁸ is hydrogen or C₁₋₄ alkyl (e.g. methyl),NHCO₂ R⁷ where R⁷ is C₁₋₆ alkyl e.g. t-butyl or SCOR⁷ where R⁷ is C₁₋₆alkyl e.g. methyl.

Alternatively, CR² R³ may represent, for example, C═O or C═NOR⁹ (whereR⁹ is a C₁₋₄ alkyl group, e.g. n-propyl).

R⁴ may particularly represent a C₁₋₄ alkoxy group such as methoxy orn-propoxy.

A particular group of compounds of the invention are compounds offormula (Ia) ##STR9## and pharmaceutically acceptable salts and solvates(e.g. hydrates) thereof, wherein R¹ to R⁴ are as defined in formula (I)above.

Preferred compounds of the invention are those compounds of formula (I)or (Ia) in which R¹ represents hydrogen or hydroxyl.

Further preferred compounds are those in which R² is in the axialconfiguration and R³ is hydrogen. Particular R² groups include C₁₋₆alkoxy, e.g. C₁₋₄ alkoxy such as methoxy or propoxy, C₁₋₄ alkoxymethoxye.g. methoxymethoxy, C₁₋₆ alkylthio (e.g. C₁₋₄ alkylthio such asmethylthio) azido, or OCOR⁷ wherein R⁷ is as defined herein above ormore particular C₅₋₈ alkyl (e.g. n-heptyl, 1-methylpentyl), phenyl,p-tolyl, 2-phenylethenyl, 1-methylethenyl or 1-methylpent-1-enyl.

Further preferred compounds are those in which R⁴ is C₁₋₄ alkoxy (e.g.methoxy or propoxy).

Particularly preferred compounds are those wherein R1 is hydrogen and R²is C₁₋₄ alkoxy or methoxymethoxy.

Further particularly preferred compounds are those wherein R¹ ishydroxyl and R² is azido, C₁₋₄ alkylthio e.g. thiomethyl, or OCOR⁷wherein R⁷ is C₄₋₈ alkyl, phenyl, tolyl, 2-phenylethenyl,1-methylethenyl or 1-methylpent-1-enyl.

It is to be understood that the present invention covers allcombinations of particular and preferred groups described hereinabove.

The compounds of formula (I) are very active fungicides useful incombatting fungal infections in animals, including humans. For example,they may be used in the treatment of fungal infections caused byorganisms such as species of Candida (e.g. Candida albicans, Candidaalabrata, (Torulorsis glabrata), Candida tropicalis, Candidaparapsilosis and Candida pseudotropicalis), Cryptococcus neoformans,Pneumocystis carinii, Asperalilius Sp (e.g. Aspergillus flavus andAsperaillus fumipatus), Coccidioides (e.g. Coccidioides immitis),Paracoccidioides (e.g. Paracoccidioides brasiliensis), Histoplasma (e.g.Histoplasma capsulatum) or Blastomyces (e.g. Blastomyces dermatitidis).They may also be used to treat other fungal infections caused by speciesof Candida, Trichophyton, Microsporum or Epidermorhyton (e.g.Trichophyton mentographytes, Trichophyton rubrum, Microsporum canis orEpidermorhyton floccosum), or in mucosal infections caused by Candidaalbicans.

Compounds of formula (I) may also be used to treat other infectionscaused by species of filamentous fungi such as Geotrichum (e.g.Geotrichum clavatum), Trichosporon (e.g. Trichosporon beigelii),Blastoschizomyces (e.g. Blastoschizomyces capitatus), Sporothrix (e.g.Sporothrix schenckii), Scedosporium (e.g. Scedosoorium apiosperum),Cladosporium (e.g. Cladosporium carrionii) and Pityrosporum ovale.

The compounds of formula (I) may also be used to treat infections causedby protozoa such as Toxoplasma, Cryptosporidium, Leishmania,Tripanosoma, Giardia and Trichomonas.

The in vitro evaluation of the anti-fungal activity of compounds of theinvention was performed on liquid or solid medium by the anti-fungaltwo-fold serial dilution technique of determining the minimum inhibitoryconcentration (MIC) of anti-fungal agent that inhibited development ofgrowth after 24 to 48 hours of incubation at 37° C. In practice, aseries of agar plates or broth microdilution panels containing two-folddilutions of anti-fungal agent tested were inoculated with a standardculture of a clinically relevant pathogen, for example, candidaalbicans. The agar plates or broth microdulution panels were thenexamined for the presence or absence of growth of the fungus and theappropriate MIC values were noted.

MFC values (defined as the lowest anti-fungal concentration that killedat least 99.9% of the initial inoculum in liquid medium) may also bedetermined by sub-culturing 0.01 and 0.1 μl of broth from the drug-freecontrol well, the first well containing growth and each clear well onagar plates.

The in vivo evaluation of compounds of formula (I) can be carried out ata series of dose levels by administration (e.g. subcutaneously, orally,intraperitoneally or intravenously) to mice or rats inoculated with astrain of Candida albicans. Untreated animals die within 3 to 9 days andthe dose level at which the test compound provides 50% protectionagainst the lethal effect of the infection is noted.

In view of their antifungal activity, compounds of formula (I) recommendthemselves for the treatment of a variety of fungal infections in humanbeings and animals. Such infections include superficial, cutaneous,subcutaneous and systemic mycotic infections such as respiratory tractinfections, gastrointestinal tract infections, cardiovascularinfections, urinary tract infections, CNS infections, candidiasis andchronic mucocandidiasis (e.g. thrush and vaginal candidiasis) and skininfections caused by fungi, cutaneous and mucocutaneous candidiasis,dermatophytoses including ringworm and tinea infections, athletes foot,paronychia, pityriasis versicolor, erythrasma, intertrigo, fungal nappyrash, candida vulvitis, candida balanitis and otitis extema. They mayalso be used as prophylactic agents to prevent systemic and topicalfungal infections. Use as prophylactic agents may, for example, beappropriate as part of a selective gut decontamination regimen in theprevention of infection in immunocompromised patients (e.g. AIDSpatients, patients receiving cancer therapy or transplant patients).Prevention of fungal overgrowth during antibiotic treatment may also bedesirable in some disease syndromes or iatrogenic states.

While it is possible that, for use in therapy, compounds of theinvention may be administered as the raw chemical, it is preferable topresent the active ingredient as a pharmaceutical formulation. Theinvention thus further provides a pharmaceutical formulation comprisingcompounds of formula (I) and physiologically acceptable salts thereoftogether with one or more pharmaceutically acceptable carriers thereofand, optionally, other therapeutic and/or prophylactic ingredients. Thecarrier(s) must be `acceptable` in the sense of being compatible withthe other ingredients of the formulation and not deleterious to therecipient thereof.

The compositions of the invention include those in a form especiallyformulated for oral, buccal, parenteral, implant, rectal, topical,ophthalmic or genito-urinary administration or in a form suitable foradministration by inhalation or insufflation.

Tablets and capsules for oral administration may contain conventionalexcipients such as binding agents, for example, syrup, acacia, gelatin,sorbitol, tragacanth, mucilage of starch or polyvinylpyrrolidone;fillers, for example, lactose, sugar, microcrystalline cellulose,maize-starch, calcium phosphate or sorbitol; lubricants, for example,magnesium stearate, stearic acid, talc, polyethylene glycol or silica;disintegrants, for example, potato starch or sodium starch glycollate orcrosscarmellose sodium; or wetting agents such as sodium laurylsulphate. The tablets which include chewable, dispersible oreffervescent tablets may be coated according to methods well known inthe art. Oral liquid preparations may be in the form of, for example,aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, ormay be presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example, sorbitolsyrup, methyl cellulose, glucoselsugar syrup, gelatin,hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gelor hydrogenated edible fats; emulsifying agents, for example, lecithin,sorbitan mono-oleate or acacia; non-aqueous vehicles (which may includeedible oils), for example, almond oil, fractionated coconut oil, oilyesters, propylene glycol or ethyl alcohol; and preservatives, forexample, methyl or propyl p-hydroxybenzoates or sorbic acid.

For buccal administration the composition may take the form of tabletsor lozenges formulated in conventional manner.

The composition according to the invention may be formulated forparenteral administration by injection or continuous infusion.Formulations for injection may be presented in unit dose form inampoules, or in multi-dose containers with an added preservative. Thecompositions may take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilising and/or dispersing agents. Alternatively theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g. sterile, pyrogen-free water, before use.

For administration by inhalation the compositions according to theinvention are conveniently delivered in the form of an aerosol spraypresentation from pressurised packs with the use of a suitablepropellant, e.g. dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas, or froma nebuliser. In the case of a pressurised aerosol the dosage unit may bedetermined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation the compositionsaccording to the invention may take the form of a dry powdercomposition, for example a powder mix of the compound and a suitablepowder base such as lactose or starch or as a modified physical form ofthe drug substance alone. The powder composition may be presented inunit dosage form in, for example, capsules or cartridges of e.g.gelatin, or blister packs from which the powder may be administered withthe aid of an inhaler or insufflator.

The compositions may take the form of a suppository, e.g. containing aconventional suppository base, or a pessary, e.g. containing aconventional pessary base.

The compositions may also be formulated for topical administration inthe form of ointments, creams, gels, lotions, shampoos, powders(including spray powders), pessaries, tampons, sprays, dips, aerosols,drops (e.g. eye, ear or nose drops) or pour-ons. Ointments and creamsmay, for example, be formulated with an aqueous or oily base with theaddition of suitable thickening and/or gelling agents. Ointments foradministration to the eye may be manufactured in a sterile manner usingsterilised components. Pour-ons may, for example, be formulated forveterinary use in oils containing organic solvents, optionally withformulatory agents, e.g. stabilising and solubilising agents. Pessariesand tampons for vaginal insertion may be formulated using conventionaltechniques and, where appropriate, may contain an effervescent vehicle.Such compositions may also contain other active ingredients such ascorticosteroids, antibiotics or antiparasitics as appropriate.

Liquid preparations for intranasal delivery may take the form ofsolutions or suspensions and may contain conventional excipients such astonicity adjusting agents, for example, sodium chloride, dextrose ormannitol; preservatives, for example benzalkonium chloride, thiomersal,phenylelklyl alcohol; and other formulating agents such as suspending,buffering, stabilising, dispersing and or flavouring agents.

Transdermal administration may be affected by the design of a suitablesystem which promotes absorption of the active compound through the skinand would typically consist of a base formulation enclosed within anadhesive stick-on patch comprising backing films, membranes and releaseliners. Such systems may include absorption enhancers such as alcoholsor work by promoting ionotophoresis.

The composition according to the invention may also be formulated as adepot preparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, a compound of the inventionmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

When the compositions comprise dosage units, each unit will preferablycontain 0.001 mg to 1000 mg, advantageously 0.01 mg to 400 mg, of activeingredient where a compound of the invention is to be administeredorally. The daily dosage as employed for adult human treatment willpreferably range from 0.001 mg to 5000 mg of active ingredient, mostpreferably from 0.01 mg to 2000 mg which may be administered in 1 to 4daily doses, for example, depending on the route of administration andon the condition of the patient and the disease to be treated.

The compound may be administered by intravenous infusion using, forexample, up to 50 mg/kg/day of the active ingredient. The duration oftreatment will be dictated by the rate of response rather than byarbitrary numbers of days.

Compounds of the invention may also be used in combination with othertherapeutic agents, and the invention thus provides, in a furtheraspect, a combination comprising a compound of the invention togetherwith another therapeutically active agent.

Thus for example the compounds of the invention may be used incombination with one or more other antifungal agents, such as a polienicderivative e.g. (Amphotericin B, Nystatin, a lipid formulation ofAmphotericin B) an azole derivative e.g. (Fluconazole, Intraconazole,Ketoconazole, Miconazole, Clotrimazole, ZD-08070, UK-109496),5-Fluorocytosine, a Pneumocandin or Echinocandin derivative such asCilofungin, LY-303366, L-733560, and/or one or more immunomodulatingagents such as an interferon e.g. (IFN-γ). interleukine e.g. (IL-1,IL-2, IL-3 and IL-8) and colony stimulating factors, [(G)-CSF, (M)-CSFand (GM)-CSF] and defensines. Particularly advantageous compounds foruse with compounds of the invention include Intraconazole, Flucytosine,Fluconazole or Amphotericin B.

When the compounds of the invention are administered in combination withanother antifungal agent the compounds of the invention and the otherfungal agent can be administered at the recommended maximum clinicaldosage or at lower doses.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with apharmaceutically acceptable carrier thereof comprise a further aspect ofthe invention. The individual components of such combinations may beadministered either sequentially or simultaneously in separate orcombined pharmaceutical formulations.

When a compound of the invention is used in combination with a secondtherapeutic agent against the same condition the dose of each compoundmay differ from that when the compound is used alone. Appropriate doseswill be readily appreciated by those skilled in the art.

According to another aspect of the present invention, we provide acompound of formula (I) or a physiologically acceptable salt thereof ora pharmaceutical composition comprising a compound of formula (I) or aphysiologically acceptable salt thereof as defined above for use intherapy, particularly for the treatment of fungal infections in animals(especially humans).

According to another aspect of the present invention, we provide the useof a compound of formula (I) or a physiologically acceptable saltthereof in the manufacture of a medicament for the treatment of fungalinfections in a human or non-human animal patient.

According to a further aspect of the present invention, we provide amethod of treatment of the human or non-human animal body to combatfungal diseases, which method comprises administering to said body aneffective amount of a compound of formula (I) or a physiologicallyacceptable salt thereof.

It will be appreciated by those skilled in the art that referencesherein to treatment extend to prophylaxis as well as the treatment ofestablished conditions or infections.

The compounds of the invention may be prepared by the processesdescribed below.

Thus, a general process (A) for the preparation of a compound offormula, (I) comprises reacting a compound of formula (II) ##STR10## (inwhich R^(1a) is as defined for R¹ in formula (I) above or is a protectedhydroxyl group, R^(4a) is as defined for R⁴ in formula (I) above or is aprotected hydroxyl group, A is a group CHO or a protected derivativethereof and R^(p) is hydrogen or a carboxyl protecting group) to replaceone or more of the free hydroxyl groups with one or more of R¹, R² /R³and R⁴, followed by the removal of any protecting groups present.

A first embodiment of process (A) comprises alkylating or alkenylating acompound of formula (II), followed by the removal of any protectinggroups present to provide a compound of formula (I) in which R² is C₁₋₁₀alkoxy, C₁₋₆ alkoxyC₁₋₄ alkoxy, arylC₁₋₆ alkyloxy, arylC₃₋₆ alkenyloxyor OR⁶ and R^(1a) and R^(4a) are as defined for R¹ and R⁴ in formula (I)above. It will be appreciated that by using appropriate alkylation andalkenylation conditions and protecting groups where necessary, one isable to selectively alkylate or alkenylate the relevant free hydroxylgroup(s) without affecting any other labile groups present in a compoundof formula (II).

The alkylation or alkenylation reaction may be effected using standardmethodology, for example by treating a compound of formula (II) with astrong base such as an alkali metal hydride (e.g. sodium hydride) in asolvent such as an ether (e.g. tetrahydrofuran), followed by theaddition of a suitable alkyl or alkenyl halide, optionally in thepresence of a tetraalkylammonium halide (e.g. tetra-n-butylammoniumfluoride or iodide). The reaction may conveniently be effected at anysuitable temperature, for example from room temperature to the refluxtemperature of the solvent.

Alternatively, the reaction may be carried out by treating a compound offormula (II) with a tin oxide (e.g. dibutyltin oxide) in a hydrocarbonsolvent (e.g. refluxing toluene), followed by the addition of analkylhalide and a fluoride salt (e.g. tetra-n-butylammonium fluoride) ina suitable solvent such as an ether (e.g. tetrahydrofuran), and heatingthe mixture in the range of about 30° to 80° C.

The introduction of a C₁₋₆ alkoxyC₁₋₆ alkyl group may conveniently beeffected using somewhat milder condition than those conditions describedabove. Thus, for example, a compound of formula (II) may be treated witha suitable alkoxyalkyl halide in the presence of a mild base such as atertiary amine (e.g. diisopropylethylamine) at a temperature in therange of about 0° to 40° C. The base may also conveniently be thesolvent for the reaction.

The introduction of a group R⁶ may conveniently be effected by treatinga compound of formula (II) with a cyclic ether containing one doublebond in the ring 2,3-position. The reaction may conveniently be effectedin a suitable solvent such as a halogenated hydrocarbon (e.g.dichloromethane) in the presence of a mild acid such as pyridiniump-toluenesulphonate at about room temperature.

According to a further embodiment of process (A), a compound of formula(I) in which R² represents a group ##STR11## may be prepared by treatinga suitably protected compound of formula (II) with an acylating agent,followed by the removal of any protecting groups present. Thus, when Xrepresents a bond or an oxygen atom, the acylation reaction may beeffected using any conventional method. For example, one method forpreparing compounds in which X is a bond comprises treating a compoundof formula (II) with a carboxylic acid R⁷ CO₂ H in the presence of anactivating agent such as dicyclohexylcarbodiimide and a suitable basesuch as 4-dimethylaminopyridine in a solvent such as a halogenatedhydrocarbon (e.g. dichloromethane) at about room temperature.Alternatively, the reaction with a carboxylic acid R⁷ CO₂ H may beeffected in the presence of 2-chloro-1-methylpyridinium iodide and asuitable base system such as 4-dimethylaminopyridine optionally alsocomprising a tertiary amine such as triethylamine, conveniently in ahalogenated hydrocarbon solvent (e.g. dichloromethane) at a temperatureof from about 20° to the reflux temperature of the solvent. An activatedacid such as a carboxylic acid halide (for example a carboxylic acidchloride R⁷ COCl) or a carboxylic anhydride such as acetic anhydride mayalso be used, whereby the acylation may be effected either by directlyreacting a compound of formula (II) with the acid halide in the presenceof a suitable base such as 4-dimethylaminopyridine, pyridine or atrialkylamine (e.g. triethylamine) or a mixture of suitable bases in asuitable solvent such as acetonitrile or dichloromethane or by additionof the acid halide (R⁷ CO)₂ O following pre-treatment of a compound offormula (II) with a tin oxide (e.g. dibutyltin oxide) in a hydrocarbonsolvent (e.g. refluxing toluene). Similar conditions using a haloformate(for example a chloroformate R⁷ OCOCl) may conveniently providecompounds in which R² represent OCO₂ R⁷.

When X represents NH, the acylation reaction may conveniently beeffected using a suitable isocyanate R⁷ NCO in the presence of a tincatalyst (e.g. a dialkyltin diacetate such as dibutyltin diacetate) andin a solvent such as an aromatic hydrocarbon (e.g. toluene) convenientlyunder reflux.

When X represents NAlkyl, the acylation reaction may conveniently beeffected using a reagent HalCONR⁷ R⁸ (where Hal is a halogen atom, e.g.chlorine) in the presence of a strong base such as an alkali metal amide(e.g. lithium diisopropylamide) and in a solvent such as an ether (e.g.tetrahydrofuran) conveniently at a temperature in the range of about 0°C. to room temperature.

In another embodiment of process (A), a compound of formula (I) in whichR¹ is hydroxyl and R² is azido or C₁₋₁₀ alkylthio may be prepared from acompound of formula (II) in which R^(1a) is hydroxyl, R^(4a) is C₁₋₆alkoxy, A is a group CHO or a protected derivative thereof and R^(p) isa carboxyl protecting group via a compound of formula (III) or (IV)##STR12## (wherein A and R^(p) are as defined just above and R⁴ is C₁₋₆alkoxy, e.g. methoxy), followed by the removal of any protecting groupspresent.

The nucleophilic ring opening of the epoxide in (III) or (IV) mayconveniently be effected by the addition of an alkali metal azide (e.g.lithium azide) or an alkali metal thioalkoxide (e.g. sodiumthiomethoxide) in a suitable solvent such as dimethylformamide. Thereaction with an azide may conveniently be effected at an elevatedtemperature, for example within the range of about 80° to 120° C. Thereaction with a thioalkoxide may conveniently be effected at about roomtemperature. It will be appreciated that the nucleophilic ring openingreaction provides compounds in which the R¹ hydroxyl group is in theopposite plane to the R² azido or C₁₋₁₀ alkylthio group. The particularconfiguration will depend upon the choice of a compound of formula (III)or (IV) as the starting material.

In a further embodiment of process (A), a compound of formula (I) inwhich R¹ and R² both represent hydrogen atoms may conveniently beprepared from a suitably protected compound of formula (II) in whichR^(1a) is a hydroxyl group by treating said compound with a reducingsystem capable of converting the 2', 3'-diol group to a 2',3'-ene groupand thereafter hydrogenating the unsaturated compound in the presence ofa palladium catalyst (e.g. palladium-on-carbon), followed, wherenecessary, by the removal of any protecting groups remaining. Theinitial reaction may conveniently be effected by treating the diol withzinc and iodine following the addition of a triarylphosphine (e.g.triphenylphosphine) and imidazole. The reaction may be carried out, forexample, in a solvent such as refluxing toluene.

Compounds of formulae (III) and (IV) may conveniently be preparedtreating a compound of formula (II) in which R^(1a) is hydroxyl, R^(4a)is C₁₋₆ alkoxy, A is a group CHO or a protected derivative hereof andR^(p) is a carboxyl protecting group to convert the free hydroxyl groupsto leaving groups such as alkyl- or arylsulphonyloxy groups followed bytreatment with a strong base such as an alkali metal hydride (e.g.sodium hydride) in a suitable solvent such as dimethylformamide,conveniently at about room temperature. Alternatively, sodium in analcoholic solvent (e.g. methanol) may be used to form the epoxide ring.In this instance the base system is added to a solution of the reactiveintermediate in a suitable solvent such as a halogenated hydrocarbon(e.g. dichloromethane) and the reaction conveniently carried out at atemperature of from about room temperature to reflux. The reactionprovides a mixture of compounds of formulae (III) and (IV) which mayconveniently be separated by flash column chromatography.

The conversion of the free hydroxyl groups in appropriate compounds offormula (II) to leaving groups may be effected using conventionalmethodology. Thus, for example, alkyl- or arylsulphonyloxy groups may beintroduced by reaction with an alkyl- or arylsulphonyl halide in thepresence of a suitable solvent such as a halogenated hydrocarbon (e.g.dichloromethane) and optionally also comprising an amine base (e.g.4-dimethylaminopyridine). The reaction may conveniently be carried outat about room temperature.

According to a further embodiment of process (A), one or more freehydroxyl groups in a compound of formula (II) may be converted to ahalogen atom using conventional displacement methods. Thus, for example,replacement with iodine may conveniently be effected by treatment withiodine in the presence of triphenylphosphine and imidazole in a suitablesolvent such as an aromatic hydrocarbon (e.g. toluene) at an elevatedtemperature, for example under reflux. Replacement with fluorine mayconveniently be effected by treatment with a suitable fluorinating agentsuch as diethylaminosulfur, trifluoride (DAST) in the presence of asolvent such as a halogenated hydrocarbon (e.g. dichloromethane) or anaromatic hydrocarbon (e.g. toluene). The reaction may conveniently beeffected about room temperature.

Another embodiment of process (A) comprises oxidising a compound offormula (II) followed by the removal of any protecting groups present toprovide a compound of formula (I) in which CR² R³ represents C═O. Theoxidation reaction may conveniently be effected using a suitableoxidising agent such as dimethylsulfoxide in the presence oftrifluoroacetic anhydride. The oxidation conveniently taken place in thepresence of a suitable solvent such as a halogenated hydrocarbon (e.g.dichloromethane), optionally comprising a suitable amine base (e.g.triethylamine), at a reduced temperature (e.g. from about -78° to 0°C.).

Another general process (B) comprises an interconversion reactionwherein a compound of formula (I) is prepared from a different compoundof formula (I) or a protected derivative thereof.

According to a first embodiment of process (B), a protected derivativeof a3 compound of formula (I) in which R² is azido may be converted to acompound of formula (I) in which R² is NR⁵ COR⁵. The R² conversion mayconveniently be effected by reducing the azido group to NH₂ byhydrogenation, for example in the presence of a suitable palladiumcatalyst (e.g. palladium on charcoal), and then acylating the amineunder conventional conditions, for example by addition of a carboxylicacid anhydride in the presence of a suitable base (e.g. pyridine),followed, when R⁵ is C₁₋₆ alkyl, by alkylation by treating the amidewith a suitable base (e.g. sodium hydride) in a solvent such as an ether(e.g. tetrahydrofuran) and then adding a suitable alkylating agent suchas an alkyl halide.

In another embodiment of process (B), a compound of formula (I) in whichR² is an azido group or the group SCOR⁷ may be prepared by treating aprotected derivative of a corresponding compound of formula (I) in whichR² is a halogen atom (e.g. iodine) with an alkali metal azide (e.g.lithium azide) or a salt e.g. sodium or cesium salt of thethiocarboxylic acid R⁷ COSH in a solvent such as dimethylformamide at anelevated temperature (e.g. 50° to 120° C.), followed by the removal ofany protecting groups present.

A further embodiment of process (B) comprises reducing a compound offormula (I) in which R² is a halogen atom (e.g. iodine) to provide,following the removal of any protecting groups present, a correspondingcompound of formula (I) in which R² is hydrogen. The reduction mayconveniently be effected using a suitable reducing agent such as a tinhydride (e.g. a trialkyltin hydride such as tributyltin hydride) in thepresence of an activating agent such as azobis (isobutyronitrile) in asolvent such as refluxing toluene.

A further embodiment of process (B) comprises alkylating a protectedderivative of a corresponding compound of formula (I) in which R¹ and/orR⁴ is hydroxyl under the conditions described in the first embodiment ofprocess (A) hereinabove.

In a further embodiment of process (B), a compound of formula (I) inwhich CR² R³ is a group C═NOR⁹ may be prepared by treating a protectedderivative of a corresponding compound of formula (I) in which CR² R³ isC═O with a reagent R⁹ ONH₂ or a salt thereof (e.g. the hydrochloridesalt) in a suitable solvent (e.g. pyridine) at an elevated temperature(e.g. from about 60° to 100° C.), followed by the removal of anyprotecting groups present.

In any of the aforementioned procedures where an alkyl moiety isintroduced as a group or part of a group, it may be appropriate to firstintroduce the corresponding unsaturated moiety and thereafter reduce thealkenyl group to the desired alkyl group, for example by hydrogenationin the presence of a palladium catalyst (e.g. 10% palladium on charcoal)at about room temperature.

Many of the above-mentioned procedures require the removal of one ormore protecting groups as a final step to provide the desired compoundof formula (I). Thus, a further general process (C) comprisesdeprotecting a protected derivative of a compound of formula (I).Suitable carboxyl protecting groups and hydroxyl protecting groups foruse herein include any conventional protecting group, for example asdescribed in "Protective Groups in Organic Chemistry", Ed. J. F. W.McOmie (Plenum Press, 1973) or "Protective Groups in Organic Synthesis"by Theodora W. Greene (John Wiley and Sons, 1991). Examples of suitablecarboxyl protecting groups include aralkyl groups such as diphenylmethyland silyl groups (e.g. trimethylsilylethyl). Examples of suitablehydroxyl protecting groups include arylalkyl groups such asp-methoxybenzyl, acyl groups such as acetyl and ester group such as2,2,2-trichloroethoxycarbonyl or benzyloxycarbonyl. Aldehyde groups mayconveniently be protected in the form of cyclic ketals.

The protecting groups may be removed using conventional techniques.Thus, a diphenylmethyl group may conveniently be removed usingtrifluoroacetic acid or by hydrogenolysis in the presence of a palladiumcatalyst (e.g. 10% palladium on charcoal). Trifluoroacetic acid mayconveniently be employed where other groups are present which aresensitive to hydrogenation. A p-methoxybenzyl group may conveniently beremoved using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Silyl groupssuch as trimethylsilylethyl may conveniently be removed using fluorideions. Cyclic ketal groups may conveniently be converted to aldehydegroup by the addition of a suitable acid such as hydrochloric acid. Anacyl group such as acetyl may conveniently be removed under basicconditions, for example using an alkoxide (e.g. sodium methoxide). Anester group such as 2,2,2-trichloroethoxycarbonyl may conveniently beremoved by adding zinc and potassium dihydrogen phosphate.

Compounds of formula (II) may conveniently be prepared from sordarin or4'-demethylsordarin, a compound of formula (V) ##STR13## Thus, forexample, compounds of formula (II) may be prepared from sordarin or4'-demethylsordarin by appropriate protection using conventional methodsas described above, followed where necessary by converting one or moreof the hydroxyl groups to other groups specified in formula (II) above.

When the carboxyl group in sordarin or a compound of formula (V) isprotected with a diphenylmethyl group the protection reaction mayconveniently be carried out by treating a solution of sordarin or acompound of formula (V) in an alcoholic solvent (e.g. methanol) or ahalogenated hydrocarbon (e.g. dichloromethane) or a mixture of suchsolvents with diphenyldiazomethane, conveniently added as a solution ina halogenated hydrocarbon solvent (e.g. dichloromethane).

When a hydroxyl group in a carboxyl protected derivative of sordarin ora compound of formula (V) is protected with a p-methoxybenzyl group thisgroup may be introduced by reaction with a tin oxide (e.g. dibutyltinoxide) in a hydrocarbon solvent (e.g. refluxing toluene), followed bythe addition of a p-methoxybenzyl halide in the presence of an fluoridesalt (e.g. tetrabutylammonium fluoride). When protected with abenzyloxycarbonyl group, this group may be introduced by reaction with abenzylhaloformate in the presence of a suitable amine base such as4-dimethylaminopyridine and in a solvent such as a halogenatedhydrocarbon (e.g. dichloromethane) or acetonitrile.

When R^(1a) in a compound of formula (II) is hydrogen such compounds mayconveniently be prepared from sordarin or 4'-demethylsordarin byappropriate protection of labile groups followed by removal of the2'-hydroxyl group. Removal of the hydroxyl group to provide a compoundin which R^(1a) is hydrogen may conveniently be effected in two stepscomprising (i) forming an S-alkyldithiocarbonate by treatment with astrong alkali metal base (e.g. sodium hydride), in the presence ofimidazole, and in a suitable solvent such as an ether (e.g.tetrahydrofuran) at a reduced temperature (e.g. about 0° C.), and thenadding carbon disulphide and an alkylhalide (e.g. methyliodide) at aboutroom temperature and (ii) removing this group by treating a solution ofthe intermediate compound in a hydrocarbon solvent (e.g. toluene) at anelevated temperature (e.g. about 80° to 120° C.) with a hydride reducingagent (e.g. a trialkyltin hydride such as tributyltin hydride),optionally in the presence of an activating agent [e.g.azobis(isobutyronitrile)].

Compounds of formulae (II), (III), (IV) and (V) are novel intermediatesand form further individual aspects of the present invention. Thecompound of formula (V), 4'-demethylsordarin, represents a particularaspect of the present invention.

Base salts of compounds of formula (I) may be conveniently formed bytreating a compound of formula (I) with an appropriate salt or base.Thus, for example, salts may conveniently be prepared by treating acompound of formula (I) with a salt or a base selected from sodium orpotassium hydroxide, hydrogen carbonate, carbonate or acetate (e.g.potassium hydroxide, potassium hydrogen carbonate, sodium hydrogencarbonate or potassium acetate), ammonium acetate, calcium acetate andL-lysine as appropriate. The salt may, for example, be prepared byadding the appropriate salt or base (if necessary as an aqueoussolution) to a solution or suspension of the compound of formula (I) ina suitable solvent such as an alcohol (e.g. methanol) or dioxane attemperatures of for example 0° C. to 80° C. and conveniently at aboutroom temperature.

Pharmaceutically acceptable salts may also be prepared from other salts,including other pharmaceutically acceptable salts of the compounds offormula (I), using conventional methods.

The novel compound of formula (V) may conveniently be prepared accordingto the fermentation process described hereinafter or by demethylatingsordarin using a biotransformation procedure. It is to be understoodthat such processes for the preparation of the compound of formula (V)represent further aspects of the present invention.

The fermentation process comprises cultivating a microorganism capableof producing the compound of formula (V) and thereafter isolating thecompound of formula (V) from the culture.

Microorganisms capable of producing the compound of formula (V) willconveniently be mutant strains of Sordaria araneosa which can beidentified by screening survivors of mutagenesis by analysing a testsample obtained from fermentation of the microorganism using standardmethodology. In particular, the microorganism to be conveniently used isa mutant strain of Sordairia araneosa deposited in the permanent culturecollection of the CAB International Mycological Institute, GeneticResource Reference Collection, Bakeham Lane, Egham, Surrey TW20 9TY,England. The strain was received by the Institute on Jun. 10, 1994 andwas subsequently given the accession number IMI 362184 and dates ofacceptance and confirmation of viability of Jun. 13 and 21 1994respectively. The Institute is an International Depository authorityrecognised under the Budapest Treaty. The characteristics thus faridentified for IMI 362184 are given in Example 46.

The present invention provides in a further aspect the microorganism IMI362184 per se and mutants thereof.

Mutants of the IMI 362184 may arise spontaneously or may be produced bya variety of methods including those outlined in Techniques for theDevelopment of Microorganisms by H I Adler in "Radiation andRadioisotopes for Industrial Microorganisms", Proceedings of theSymposium, Vienna 1973, p241, International Atomic Energy authority.Such methods include ionising radiation, chemical methods, e.g.treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), heat, genetictechniques, such as recombination and transformation, and selectivetechniques for spontaneous mutants.

The preparation of the compound of formula (V) by fermentation may beeffected by conventional means i.e. by culturing a suitable organism inthe presence of assimilable sources of carbon, nitrogen and mineralsalts, and thereafter isolating the desired product.

Assimilable sources of carbon, nitrogen and minerals may be provided byeither simple or complex nutrients. Sources of carbon will generallyinclude glucose, maltose, starch, glycerol, molasses, dextrin, lactose,sucrose, fructose, galactose, myo-inositol, D-mannitol, soya bean oil,carboxylic acids, amino acids, glycerides, alcohols, alkanes andvegetable oils. Sources of carbon will generally comprise from 0.5 to10% by weight of the fermentation medium. Fructose, glucose and sucroserepresent preferred sources of carbon.

Sources of nitrogen will generally include soya bean meal, corn steepliquors, distillers solubles, yeast extracts, cottonseed meal, peptones,ground nut meal, malt extract, molasses, casein, amino acid mixtures,ammonia (gas or solution), ammonium salts or nitrates. Urea and otheramides may also be used. Sources of nitrogen will generally comprisefrom 0.1 to 10% by weight of the fermentation medium.

Nutrient mineral salts which may be incorporated into the culture mediuminclude the generally used salts capable of yielding sodium, potassium,ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium,chromium, calcium, copper, molybdenum, boron, phosphate, sulphate,chloride and carbonate ions.

Cultivation of the organism will generally be effected at a temperatureof from 20° to 40° C., preferably from 20 to 35° C., especially about25° C., and will desirably take place with aeration and agitation e.g.by shaking or stirring. The medium may initially be inoculated with asmall quantity of mycelium and/or spores. The vegetative inoculumobtained may be transferred to the fermentation medium, or to one ormore seed stages where further growth takes place before transfer to theprincipal fermentation medium. The fermentation will generally becarried out in the pH range 3.5 to 9.5, preferably 4.5 to 7.5. It may benecessary to add a base or an acid to the fermentation medium to keepthe pH to within the desired range. Suitable bases which may be addedinclude alkali metal hydroxides such as aqueous sodium hydroxide orpotassium hydroxide. Suitable acids include mineral acids such ashydrochloric, sulphuric or phosphoric acid.

The fermentation may be carried out for a period of 4-30 days,preferably about 5-15 days. An antifoam may be present to controlexcessive foaming and added at intervals as required. Carbon and/ornitrogen sources may also be fed into the fermentation medium asrequired.

The compound of formula (V) is associated mainly with the cells and maybe brought into solution either by addition of an acid and awater-miscible organic solvent, or more preferably by addition of a base(e.g. sodium hydroxide). Cells may be separated from these solutionseither by centrifugation, conventional filtration or membranefiltration. The liquor may be optionally thereafter treated with an acidsuch as sulphuric acid until the pH is below 6 (e.g. about pH 4.5).

The compound of formula (V) may be isolated and purified by a variety offractionation techniques, for example adsorption-elution, precipitation,fractional crystallisation, solvent extraction and liquid-liquidpartition which may be combined in various ways.

Adsorption onto a solid support followed by elution has been found to beparticularly suitable for isolating and purifying the compound offormula (V).

Suitable solid supports include silica; a non-functional macroreticularadsorption resin, for example cross-linked styrene divinyl benzenepolymer resins such as CG161 and Amberlite XAD-2, XAD4, XAD-16 orXAD-1180 resins (Rohm & Haas Limited) or Kastell S112 (Montedison); asubstituted styrene-divinyl benzene polymer such as Diaion SP207(Mitsubishi); an anion exchanger [e.g. IRA-958 or MacroPrep High Q(BioRad)], an organic solvent-compatible cross-linked dextran such asSephadex LH20 (Pharmacia UK Limited), or on reverse phase supports suchas hydrocarbon linked silica, e.g. C₁₈ -linked silica.

The compound of formula (V) may also be isolated and purified by the useof a liquid anion exchanger such as LA 2.

Suitable solvents for the elution of the compound of formula (V) will,of course, depend on the nature of the absorbent. When using a polymerresin such as XAD-16 water-miscible solvents such as methanol, acetone,isopropanol or acetronitrile in various proportions in water may beparticularly suitable.

The presence of the compound of formula (V) during theextraction/isolation procedures may be monitored by conventionaltechniques such as high performance liquid chromatography (HPLC) or UVspectroscopy or by utilising the optical rotation or other property ofthe compound.

Where the compound of formula (V) is obtained in the form of a solutionin an organic solvent, for example after purification byabsorption/elution, the solvent may be removed by conventionalprocedures, e.g. by evaporation, to yield the required compound. Ifdesired, the compound may be further purified by chromatographictechniques such as countercurrent chromatography using a coil extractersuch as a multi-layer coil extracter or high performance liquidchromatography or supercritical fluid chromatography on adsorbents suchas carbon, alumina, vanadium, polymeric resins or silica, with orwithout bonded phases. Suitable solvents/eluents for the chromatographicpurification/separation of the compound of formula (V) will of coursedepend on the nature of the adsorbent. When using a C8 bonded silica,mixtures of acetonitrile and water are particularly suitable.Alternatively, the compound may be further purified by solventextraction, for example using an appropriate organic solvent such as aketone (e.g. acetone or methyl ethyl ketone), a halogenated hydrocarbon,an alcohol (e.g. methanol), a diol (e.g. propane-1,2-diol orbutane-1,3-diol) or an ester (e.g. methyl acetate or ethyl acetate). Ina further alternative, solutions of compound (V) may be further purifiedby treatment with adsorbents that selectively remove impurities whenadded at appropriate levels (e.g. DEAE-cellulose) or by crystallisation(e.g. from a mixture of acetonitrile and water) or using a combinationof the above procedures.

The biotransformation of sordarin to 4'-demethylsordarin, the compoundof formula (V), may be effected by incubating sordarin in a culturecomprising a suitable organism and sources of carbon and nitrogen,including those sources specified hereinabove, and thereafter isolatingthe compound of formula (V) from the culture.

Microorganisms capable of demethylating sordarin at the 4'-position mayreadily be identified by using a small scale test and analysing a testsample obtained using standard methodology, for example, using HPLC.Examples of microorganisms which have been identified as sordarindemethylators include Streptomyces capreolus ATCC 31963, Streptomycesavermitilis ATCC 31272, Streptomyces armentosus NRRL 3176, Streptomycesantibioticus ATCC 31771, Streptomyces rimosus ATCC 23955, Streptomycesplatensis ATCC 29778, Streptomyces mashuensis ATCC 23934, Streptomyceseurythermus ATCC 14975, Nocardia orientalis ATCC 43491 andCunninghamella echinulata var elegans ATCC 36112.

Cultivation of the organism will generally be effected at a temperatureof from 20° to 40° C., preferably from 20° to 35° C., especially about28° C., and will desirably take place with aeration and agitation, e.g.by shaking or stirring. The medium may initially be inoculated with asmall quantity of mycelium and/or spores. The vegetative inoculumobtained may be transferred to the fermentation medium or to one or moreseed stages where further growth (e.g. over about 1-3 days) takes placebefore transfer to the principal fermentation medium. The principalfermentation medium will also comprise sordarin and the fermentationwill generally be carried out in a pH range of 3.5 to 9.5, preferably4.5 to 7.5. It may be necessary to add a base or an acid to thefermentation medium to keep the pH to within the desired range. Suitablebases which may be added include alkali metal hydroxides such as aqueoussodium hydroxide or potassium hydroxide. Suitable acids include mineralacids such as hydrochloric, sulphuric or phosphoric acid. Fermentationmay be carried out over a period of 2 to 5 days, preferably about 3days. An antifoam may be present to control excess foaming and added atintervals as required. Carbon and/or nitrogen sources may also be fedinto the fermentation medium as required.

The separation and isolation of the compound of formula (V) from thefermentation broth may be effected by the general procedures previouslydescribed. When it is desired to lower the pH of the liquor to below pH6 (e.g. to about pH 2.5) this may conveniently be achieved by theaddition of an acid such as orthophosphoric acid.

It will be appreciated that biotransformation may be effected accordingto a number of alternative methods. For example, cells may be grown andharvested prior to addition to a solution of sordarin in, for example,buffer, spent fermentation medium or water. It is also feasible that theappropriate enzymes could be isolated and used (with appropriateco-enzymes) or the enzymes cloned and over-expressed.

As stated hereinabove, sordarin is a known compound, which may beobtained using procedures described in the relevant art. Thus, forexample, the preparation of sordarin by the cultivation of Sordariaaraneosa NRRL 3196 (also deposited in the ATCC as ATCC 36386) isdescribed in British Patent Specification No. 1,162,027. Specificexamples of the preparation of sordarin using similar procedures arereported hereinafter.

Sordaricin may conveniently be prepared under fermentation conditionssimilar to those described for preparing sordarin using Sordariaaraneosa NRRL 3196 or a suitable mutant thereof, with isolation of thedesired compound using appropriate chromatographic means. One suchmutant has been deposited in the permanent culture collection of the CABInternational Mycological Institute, Genetic Resource ReferenceCollection, Bakeham Lane, Egham, Surrey TW20 9TY, England. The strainwas received and accepted by the Institute on Aug. 11, 1994 and wassubsequently given the accession number IMI 362947 and a date ofconfirmation of viability of Aug. 19, 1994. The Institute is anInternational Depository authority recognised under the Budapest Treaty.The characteristics thus far identified for IMI 362947 are given inExample 47.

The present invention provides in a further aspect the microorganism IMI362947 per se and mutants therof.

Processes for obtaining mutants of IMI 362947 and its genetic materialwill be similar to those described hereinabove for the manipulation ofIMI 362184.

Sordaricin may also be prepared from sordarin using a biotransformationprocedure. The biotransformation may conveniently be effected byincubating sordarin in a culture comprising a suitable organism andsources of carbon and nitrogen, including those sources specifiedhereinabove, and thereafter isolating sordaricin from the culture.

Microorganisms capable of converting sordarin to sordaricin may readilybe identified by using a small scale test and analysing a test sampleobtained using standard methodology, for example, using HPLC. We haveidentified one such microorganism and deposited it with the NationalCollections of Industrial and Marine Bacteria Limited (NCIMB), 23 St.Machar Drive, Aberdeen AB2 1RY, Scotland. The strain was received by theNCIMB on Aug. 4, 1994 and was the same day accepted for deposit forpatent purposes and the viability of the microorganism confirmed. Themicroorganism, which is a Coryneform species having the characteristicsgiven in Example 48, has been assigned the accession number NCIMB 40675.The NCIMB is an International Depository authority recognised under theBudapest Treaty.

The invention thus provides in another aspect the microorganism NCIMB40675 per se and mutants thereof.

According to another aspect of the present invention we provide thegenetic material of NCIMB 40675 and mutants thereof that participates inthe bioconversion of sordarin to sordaricin.

Processes for obtaining mutants of NCIMB 40675 and its genetic materialwill be similar to those described hereinabove for the manipulation ofIMI 362184.

Cultivation of the NCIMB 40675 will generally be effected at atemperature of from 20° to 40° C., preferably from 20° to 35° C.,especially about 28° C., and will desirably take place with aeration andagitation, e.g. by shaking or stirring. The medium may initially beinoculated with a small quantity of mycelium and/or spores. Thevegetative inoculum obtained may be transferred to the fermentationmedium or to one or more seed stages where further growth (e.g. overabout 1-3 days) takes place before transfer to the principalfermentation medium. The principal fermentation medium will alsocomprise sordarin and the fermentation will generally be carried out ina pH range of 3.5 to 9.5, preferably about 7.5. It may be necessary toadd a base or an acid to the fermentation medium to keep the pH towithin the desired range. Suitable bases which may be added includealkali metal hydroxides such as aqueous sodium hydroxide or potassiumhydroxide. Suitable acids include mineral acids such as hydrochloric,sulphuric or phosphoric acid. Fermentation may be carried out over aperiod of 4 to 8 days, preferably about 6 days. An antifoam may bepresent to control excess foaming and added at intervals as required.Carbon and/or nitrogen sources may also be fed into the fermentationmedium as required.

It will be appreciated that biotransformation may be effected accordingto a number of alternative methods. For example cells may be grown andharvested prior to addition to a solution of sordarin in, for example,buffer, spent fermentation medium or water. It is also feasible that theappropriate enzymes could be isolated and used or the enzymes cloned andoverexpressed.

The separation and isolation of sordaricin from the fermentation brothmay be effected by the general procedures previously described. When itis desired to lower the pH of the liquor to about pH 6 this mayconveniently be achieved by the addition of an acid such asorthophosphoric acid.

It is to be understood that the fermentation and bioconversion processesdescribed hereinabove for preparing sordaricin represent further aspectsof the present invention.

The examples hereinafter illustrate aspects of the present invention andare not intended to limit the invention in any way.

PREPARATION 1

Production of sordarin

A culture of Sordaria araneosa NRRL3196 (ATCC36386) was grown on an agarmedium until mature growth occurred. 6 mm diameter plugs of the agarcontaining the growth were transferred to sterile water or Brain HeartInfusion broth (Oxoid)+10% glycerol and stored at ambient temperature or-140° C. respectively. A suspension containing 2 of these agar plugs wasused to inoculate a 250 ml Erlenmyer flask containing 50 ml of mediumFS.

    ______________________________________                                        Medium FS            g/L                                                      ______________________________________                                        Peptone(Oxoid L34)   10                                                         Malt extract (Oxoid L39)        21                                            Glycerol (Glycerine CP)         40                                            Junlon 110 (Honeywell & Stein)   1                                            Distilled water                                                             ______________________________________                                    

The culture was incubated at 25° C. for 5 days on a rotary shakeroperated at 250 rpm with a 50 mm diameter orbital motion. Aliquots (2ml) of the developed inoculum were used to inoculate further 250 mlErlenmyer flasks containing medium FS (50 ml) which were incubated asdescribed above. 80 ml of the bulked shake flask developed inoculum wasused to inoculate each of two 7L fermenters containing 5L of medium FS.The fermentations were controlled to a temperature of 25C. The culturewas agitated at 400 rpm and aerated at 2 Lpm. After 3 days fermentation,10L of culture was used to inoculate a 780L fermenter containing 500L ofmedium SM55VAR.

    ______________________________________                                        SM55VAR              g/L                                                      ______________________________________                                        Glucidex 32D (Roquette Frere)                                                                      74                                                         Peptone (Oxoid L37)            10                                             Proflo (Traders Protein)       30                                             Beet molasses                  15                                             MgSO.sub.4.7H.sub.2 O(BDH)                5                                   CaCO.sub.3 (BDH)                    5                                         FeSO.sub.4.7H.sub.2 O(Sigma)             2                                    ZnSO.sub.4.7H.sub.2 O(BDH)               0.04                                 L-trytophan(Sigma)            2                                               PPG2000 (K & K Greef)          0.5                                            Silicone 1520 (Dow Corning)    0.04                                           Distilled water                                                             ______________________________________                                    

The fermentation was controlled to a temperature of 25° C. The broth wasagitated at 300-350 rpm and aerated at 200 Lpm. 70% (w/v) Meritose(Tunnel Refineries) solution was fed to the culture to maintain apositive residual glucose concentration. Distilled water was fed tomaintain a culture volume of 500L. PPG2000 antifoam was added on demandto control foaming. Whole broth extracts (in aqueous acetonitrile +1%trifluoroacetic acid) were assayed for presence of sordarin by reversephase HPLC. The culture was harvested after 11 days when the extract ofa broth sample indicated a sordarin titre of 0.6 g/L. Fermentation brothwas made 0.1M with respect to sodium hydroxide and after storage atambient temperature overnight was filtered through Dicalite on a rotaryvacuum filter (1% Dicalite was added to the broth as filter aid). Thefiltrate was adjusted to pH 6-7 with concentrated sulphuric acid and thesolution was applied to XAD16 resin (10 volumes of filtrate/volumeresin). The adsorbent was washed with water and acetone:water (1:3) inboth cases to give a clear effluent before sordarin was eluted withacetone:water (3:1; 2 column volumes collected). Flow rate throughoutthe process was between 1-2 column volumes/hr. The eluate wasconcentrated to a small volume (8.5L). The concentrate was adjusted topH 3 with phosphoric acid and stood overnight at ambient temperature toallow precipitated sordarin to settle. Supernatant was decanted thencentrifuged and the supernatant was discarded. Centrifuge pellets andprecipitate were taken up in 75% aqueous acetonitrile to give 3.9L of adark brown solution. To this was added 1.0L 0.2M NH₄ H₂ PO₄ withstirring, and the solution was adjusted to pH 4.0 with phosphoric acid,to give a final volume of 5.0L with approximate composition 60%acetonitrile -0.1M NH₄ H₂ PO₄. The crude sordarin solution (5.0L) fromabove was subjected to preparative HPLC in 10 injections (450-550 mleach) on a column (15 cm ×25 cm) of 7 mm Kromasil C8 in mobile phase of50% acetonitrile -0.1M NH₄ H₂ PO4, pH 4 (50L acetonitrile made up to 100L with water containing 575 g NH₄ H₂ PO₄ and 40 ml H₃ PO₄), flow-rate600ml/min, detection by UV absorbance (λ210 nm). The fraction elutingbetween 15.4 and 19.2 min was collected. Pooled fractions from the 10injections (23L) were diluted with water to 50L and this solution waspumped back through the Kromasil column at 28L/h. The column was washedwith water (25L) then eluted with 90% acetonitrile (10L). The eluate wasevaporated to a residue of 1300 ml which was freeze-dried to yield thetitle compound as a buff powder (105.6 g). MS and NMR analysis of theproduct showed equivalence with an authentic sample of sordarin.

PREPARATION 2

Production of sordarin potassium salt

Sordaria araneosa NRRL3196 (ATCC36386) was maintained in Brain HeartInfusion broth (Oxoid)+10% glycerol at -140° C. as described inPreparation 1. A suspension containing 2 agar plugs was used toinoculate a 250 ml Erlenmyer flask containing 50 ml of medium FS. Theculture was incubated at 25° C. for 5 days on a rotary shaker operatedat 250 rpm with a 50 mm diameter orbital motion. Aliquots (2 ml) of thedeveloped inoculum were used to inoculate further 250 ml Erlenmyerflasks containing medium FS (50 ml) and incubated as described above. 80ml of the bulked shake flask developed inoculum was used to inoculateeach of two 7L fermenters containing 5L of medium FS. The fermentationswere controlled to a temperature of 25° C. The culture was agitated at400 rpm and aerated at 2 Lpm. After 3 days fermentation, 10L of culturewas used to inoculate a 780L fermenter containing 500L of medium

    ______________________________________                                        SD1                  g/L                                                      ______________________________________                                        Meritose (Tunnel refineries)                                                                       22                                                         Lactose                         20                                            Glucidex 32D (Roquette Frere)   30                                            Arkasoy (The British Arkady Co.) 20                                           CSL                             20                                            FeCl.sub.3.6H.sub.2 O                      0.05                               NH.sub.4 H.sub.2 PO.sub.4                        5                            ZnSO.sub.4.7H.sub.2 O(BDH)                0.1                                 PPG2000                         0.5                                           Distilled water                                                             ______________________________________                                    

The fermentation was controlled to a temperature of 25° C. The broth wasagitated at 350 rpm and aerated at 200 Lpm. Distilled water was fed tomaintain a culture volume of 500L. Whole broth extracts were assayed forpresence of sordarin by reverse phase HPLC. The culture was harvestedafter 6 days when the extract of a broth sample indicated a sordarintitre of 1.3 g/L. A 50L sample of harvest broth was made 0.1M withrespect to sodium hydroxide and stored at 4° C. overnight. Cells wereremoved by vacuum filtration through a bed of Dicalite adding anadditional 2% Dicalite to the broth as filter aid. The filtrate wasadjusted to approximately pH 6 with concentrated sulphuric acid. Brothextract was pumped through a bed of Amberchrom CG161 resin at 320 ml/min(0.64 bed volumes per min). The effluent was monitored by HPLC. After 45min (equivalent to a pumped volume of 14.4L) sordarin began to breakthrough and pumping was halted. The adsorbent was washed with water (2L)then 25% v/v acetone in water (2L). Sordarin was eluted with 75% v/vacetone in water (1.5L). The 75% v/v acetone eluate was evaporated toabout 200 ml by rotary evaporation at 40° C. 200 ml butan-1-ol was addedand the evaporation continued until a viscous oil remained (containingsome butan-1-ol). The oily residue was extracted with hot methanol(2×500 ml). Extracts were combined, filtered (Whatman no 1 paper) thenevaporated at 40 to 45° C. to give a viscous oil. This was extractedwith hot acetone (2×500 ml). Acetone extracts were combined, filteredand evaporated to a viscous oil. Propan-2-ol (350 ml) was added and theoil dissolved at 45° C. to give a clear brown solution. A solution ofpotassium-2-ethylhexanoate (39% wow solution in propan-2-ol, 54 g) wasweighed into a 500 ml conical flask. The sordarin-containing solution inpropan-2-ol was poured into the conical flask, the contents mixed, andleft to stand for 4h at room temperature. The solution was seeded withsordarin potassium salt (about 5 mg) and the stoppered flask was storedfor 3 days at 4° C. The off-white solid which formed was filtered undervacuum (No 4 sinter funnel) and the filter cake was washed withpropan-2-ol (about 20-30 ml). The solid was transferred to acrystallising dish and dried in vacuo over P₂ O₅ for 16h to give thetitled compound (10.5 g).

PREPARATION 3

Production of sordarin potassium salt

500 litres of culture broth was prepared as in Preparation 1. The brothwas made 0.1M with respect to sodium hydroxide and after 4 days at 0° C.was filtered through a bed of Dicalite on a rotary vacuum filter. 1%Dicalite was added to the broth as filter aid. The pH of the filtratewas adjusted from pH 9.16 to pH 7.5 with concentrated sulphuric acid.Filtrate (10L) was adjusted to pH 6 with H₃ PO₄ and applied to a bed(200 ml) of XAD-16 resin packed in water at a flow-rate of 1-1.5 bedvolumes/h. The resin bed was washed with water (1 bed volume) then with25% aqueous isopropanol (2.5 bed volumes) before elution with neatisopropanol. After a fore-run of 50 ml the isopropanol eluate wascollected as 100 ml fractions. Sordarin-rich fractions 2-6 inclusivewere pooled and evaporated to half volume. Isopropanol was added tobring the volume back to 500 ml, then the evaporation to half volumerepeated to remove residual water by azeotropic distillation. After athird evaporation to half volume the residue was filtered and the filterwas washed with isopropanol. To the combined filtrate and washings (400ml) was added a solution of potassium 2-ethylhexanoate (8 g) inisopropanol (100 ml). The mixture was seeded with sordarin potassiumsalt and stood for several days at 4° C. whilst a slow crystallisationtook place. Crystals were filtered on a No. 3 sinter, washed with alittle ice-cold isopropanol and dried in vacuo to yield the titlecompound as a pale brown powder (4.85 g).

PREPARATION 4

Production of 4'-demethylsordarin

(i) IMI 362184 was maintained in Brain Heart Infusion broth (Oxoid)+10%glycerol at -140° C. as described in Preparation 1. A suspensioncontaining 2 agar plugs was used to inoculate a 250 ml Erlenmyer flashcontaining 50 ml of medium FS. The culture was incubated at 25° C. for 5days on a rotary shaker operated at 250 rpm with a 5 mm diameter orbitalmotion. Aliquots (2 ml) of the developed inoculum were used to inoculatefurther 250 ml Erlenmyer flasks containing medium FS (50 ml) andincubated as described above. 80 ml of the bulked shake flash developedinoculum was used to inoculate each of two 7L fermenters containing 5Lof medium FS. The fermentations were controlled to a temperature of 25°C. The culture was agitated at 400 rpm and aerated at 2 Lpm. After 3days fermentation, 10L of culture was used to inoculate a 780L fermentercontaining 500L of medium SM55VAR (as described in Preparation 1). Thefermentation was controlled to a temperature of 25° C. The broth wasagitated at 350 rpm and aerated at 500 Lpm. 70% w/v Meritose (TunnelRefineries) solution was fed to the culture to maintain a positiveresidual glucose concentration. Distilled water was fed to maintain aculture volume of 500L. Whole broth extracts were assayed for presenceof 4'-demethylsordarin by reverse phase HPLC. The culture was harvestedafter 10 days when the extract of a broth sample indicated a4'-demethylsordarin titre of 0.8 g/L. Fermentation broth was made 0.1Mwith respect to sodium hydroxide and after 1 hour at ambient temperaturewas filtered through Dicalite on a rotary vacuum filter (1% Dicalite wasadded to the broth as filter aid). The filtrate was adjusted to pH 4.5with concentrated sulphuric acid and the solution was applied to XAD16resin (20 g product/L resin). The adsorbent was washed with 0.1%phosphoric acid (10 column volumes) and acetonitrile:water 1:4 (6 columnvolumes) before the product was eluted with acetonitrile:water (1:1; 2column volumes). Flow rate throughout the process was between 1-2 columnvolumes/hr. The eluate was concentrated to dryness with the addition ofbutan-1-ol and the solid was extracted with methanol (12L) followed byacetone (10L) at 60° C. Insoluble material was removed at each stage byfiltration through a no 3 glass sinter and the extracts concentrated todryness as before. The solid was crystallised from acetonitrile:water(3:7) before being recrystallised from the same solvent and dried toconstant weight over P₂ O₅ to give the title compound (244.0 g), whichby proton NMR analysis showed equivalence with an authentic sample of4'-demethylsordarin.

(ii) The fermentation procedure in (i) above was followed, and after thebroth was made 0.1M with respect to sodium hydroxide this wasultrafiltered through ETNA 10A membrane (10 kDalton cut-off). Afterdiafiltration with water the bulked permeate was a clear solution. Afteradjustment to pH 5.2, using concentrated sulphuric acid, the permeatewas loaded to a column of XAD16 resin at a rate of 2 column volumes perhour to give a final loading of 32 g 4'-demethylsordarin per litre ofresin. The column was washed at a rate of 2 column volumes per hour,first with 0.1% v/v phosphoric acid and then with 20% v/vacetonitrile/water (10 column volumes of each). The column was elutedwith 65% acetonitrile/water at 2 column volumes per hour. A forerun of0.75 column volumes was discarded. 85% of the loaded 4'-demethylsordarinwas recovered in the next 1.6 column volumes of eluate. The rich eluatewas treated by stirring for 5 minutes with 2% w/v of DE52 cellulose,which was removed by filtration. An aliquot of DE52 treated eluate wasconcentrated to 62% of the original volume (43% acetonitrile) using arotary evaporator. The concentrated eluate was held at 4° C. for 15hours to crystallise and the solids formed collected by filtrationthrough a glass sinter. The crystals were washed with four cake volumesof 30% v/v acetonitrile/water and dried at 30° C. in vacuo. The titlecompound (1.94 g) was recovered from the concentrated eluate as a palegrey solid.

PREPARATION 5

Screen for microorganisms capable of demethylating sordarin at the 4'position

Microorganisms capable of demethylating sordarin at the 4'-positioncould be identified by growing them at 28° C. (bacteria) or 25° C.(fungi) in 10 ml volumes of SB1 (bacteria) or FB1 (fungi) in 50 mlconical flasks shaken at 250 rpm. After 2 days, sordarin was added to afinal concentration of 0.5 mg/ml (from a 200 mg/ml stock solution in 80%ethanol) and the flasks were then incubated for a further 3 days. A 500μl sample of whole culture was mixed in an Eppendorf tube with 500 μl of80% acetonitrile/2% trifluoroacetic acid and left to extract at roomtemperature for 30 minutes. The extract supernatant, obtained bycentrifuging samples in a microfuge was assayed by isocratic HPLC forthe presence of 4'-demethylsordarin. 4'-demethyl sordarin eluted at 3.35mins with a mobile phase of 35% acetonitrile in water, flow rate 2ml/min, using a Spherisorb C₆ column (5 μm, 15 cm ×4.6 mm). By thismethod, the following microorganisms were identified as sordarindemethylators:

    ______________________________________                                        Streptomyces capreolus  ATCC 31963                                              Streptomyces avermitilis                   ATCC 31272                         Streptomyces armentosus                    NRRL 3176                          Streptomyces antibioticus                 ATCC 31771                          Streptomyces rimosus                        ATCC 23955                        Streptomcyes platensis                     ATCC 29778                         Streptomyces mashuensis                    ATCC 23934                         Streptomyces eurythermus                   ATCC 14975                         Nocardia orientalis                       ATCC 43491                          Cunninghamella echinulata var elegans      ATCC 36112                       ______________________________________                                                        g/L                                                           ______________________________________                                          SB1 Medium                                                                    Arkasoy                       25                                              Yeast extract                   5                                             KH.sub.2 PO.sub.4                  5                                          Glucose                       20                                              Distilled Water                                                               pH 7                                                                          FB1 Medium                                                                    Soya oil                     30                                               Arkasoy                     10                                                Yeast extract                5                                                K.sub.2 HPO.sub.4               5                                             Glucose                     20                                                Distilled Water                                                               pH 5.5                                                                      ______________________________________                                    

PREPARATION 6

Production of 4'-demethylsordarin by biotransformation of sordarin

0.3ml of a spore suspension of Streptomyces capreolus ATCC 31963 (in15%, v/v glycerol stored at -20° C.) was inoculated into 30 ml SB1medium in a 250 ml Erlenmeyer flask to give a seed culture which wasincubated at 28° C. and 250 rpm on a rotary shaker. After 4 days, 0.5 mlof this was used to inoculator 35 ml SB1 in a 250 ml flask which wasgrown for 48 hours at 28° C., 250 rpm. At this stage, the culture wasaliquoted as 10 ml amounts into 50 ml Erlenmeyer flasks which were fedwith 5 mg sordarin (from a 200 mg/ml stock solution in ethanol).Incubation was continued for a further 3 days. 80% v/v acetonitrile inwater (14 ml) was added to whole broth (14 ml) and the mixture was keptat room temperature and occasionally agitated. After 30 minutes, thecells were removed by centrifugation. Acetonitrile was removed byevaporation and the pH of the aqueous solution was adjusted to 2.5 withorthophosphoric acid. The solution was passed through a columncontaining Amberlite XAD-16 resin (bed volume 5 ml). The adsorbent waswashed sequentially with water (10 ml), 10% v/v acetonitrile in water(20 ml), 30% v/v acetonitrile in water (10 ml), 50% v/v acetonitrile inwater (20 ml) and 90% v/v acetonitrile in water (10 ml). Fractions weremonitored by HPLC; 4'-demethylsordarin was located in the 50% v/vacetonitrile in water eluate. The fraction containing4'-demethylsordarin was evaporated to dryness in vacuo at the roomtemperature and the residue was re-dissolved in 35% v/v acetonitrile inwater (15 ml). 4'-demethylsordarin was purified by preparative HPLC:

    ______________________________________                                        Column    Spherisorb 5 micron C6 25 cm ×  2.5 cm                          Flow rate         25 ml/min                                                   Detection        UV at 210 nm                                                 Mobile Phase     350 ml acetonitrile made up to 1000 ml with 0.05M                          ammonium dihydrogen phosphate in water. pH                           adjusted to 2.5 with orthophosphoric acid                                Injection volume 4.5 ml                                                     ______________________________________                                    

4'-Demethylsordarin was eluted after 10.0 minutes under theseconditions. The pooled fractions from four HPLC runs were diluted 1:1with water then pumped back onto the silica (after washing this withacetonitrile and re-equilibrating with water). The adsorbent was washedwith water (200 ml) and adsorbed product was eluted with 95% v/vacetonitrile in water (200 ml). The acetonitrile/water eluate wasevaporated to remove organic solvent, and the aqueous solution freezedried to yield 4'-demethylsordarin (1.5 mg) as a white powder.

δ (¹ H,CDCl₃); 9.74(s,1H); 6.08(brd,3,1H); 4.70(d,1.5,1H);4.16(d,9.5,1H); 4.08(dd,4.5,3.5,1H); 3.88(dd,4.5,1.5,1H);3.75(dq,8.5,6,1H); 3.62(d,9.5,1H); 3.68(dd,8.5,3.5,1H); 2.65(m,1H);2.34(m,1H); 1.32(d,6,3H); 1.30(d,12.5,1H); 1.23(m,1H); 1.04(d,7,3H);0.99(d,7,3H); 0.81(d,7,3H)

INTERMEDIATE 1

[1R-(1α,3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a[(6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of sordarin (10.0 g) in dichloromethane (150 ml) was treateddropwise with a solution of diphenyidiazomethane in dichloromethane(0.35M, 85 ml). The resulting solution was stirred at room temperaturefor 24 hours. The solvent was removed under reduced pressure and theresidue was purified by flash column chromatography on silica geleluting with hexane:ethyl acetate (4:1) and (2:1). The fractions werecombined and evaporated to yield the title compound (11.8 g) as a whitefoam.

δ (¹ H, CDCl₃): 10.00 (s, 1H, CHO), 7.63 (m, 10H, 2Ph), 7.26 (s, 1H,CHPh₂), 6.30 (dd, 1H, H-2, J=1.2 and 3.3 Hz), 4.92 (d, 1H, H-1', J=0.9Hz), 4.84 (t, 1H, H-3', J=3.3 Hz),4.03 and 4.35 (2d, 2H, 8aCH₂, J=9 Hz),3.88 (m, 1H, H-2'), 3.70 (dq, 1H, H-5', J=6.3 and 9.3 Hz), 3.41 (s, 3H,4'--OMe), 3.20 (dd, 1H, H4', J=3.3 and 9 Hz), 2.75 (t, 1H, H-1).

INTERMEDIATE 2

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-Benzyl-6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of Intermediate 1 (0.6 mmol) in dry tetrahydrofuran (15 ml)under nitrogen was treated with sodium hydride (1.5 mmol). After 30minutes, a solution of benzyl bromide (0.7 mmol) in dry tetrahydrofuran(5 ml) was added. The mixture was stirred under reflux for 4.5 hours andthen poured into water:ethyl acetate (1:1;100 ml). The organic phase wasevaporated and the residue purified by flash chromatography usinghexane:ethyl acetate (3:1) as eluent. Appropriate fractions wereconcentrated to give the title compound (249 mg).

δ (¹ H, CDCl₃): 9.76 (s, 1H, CHO), 7.33 (m, 15H, 3×Ph), 6.99 (s, 1H,CHPh₂), 6.03 (dd, 1H, H-2, J=1.2 and 3.3 Hz), 4.84 and 4.64 (AB system,1H, 1H, CHPh₂, J=12.6 Hz), 4.62 (d, 1H, H-1', J=1.2 Hz), 4.12 (m, 2H,H-3' and 8a-CHa), 3.66 (m, 3H, H-2', H-5' and 8a-CHb), 3.21 (dd, 1H,H4', J=3.3 and 9.3 Hz), 2.70 (t, 1H, H-1, J=3.9 Hz), 2.92 (d, 1H, OH,J=9.9 Hz), 2.21 (m, 1H, CH(CH₃)₂).

INTERMEDIATE 3

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-3-O-hexyl-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of Intermediate 1 (0.6 mmol) in dry tetrahydrofuran (15 ml)under nitrogen was treated with sodium hydride (1.5 mmol). After 30minutes, a solution of hexyl iodide (0.7 mmol) in dry tetrahydrofuran (5ml) was added. The mixture was stirred under reflux for 36 hours andthen poured into water:ethyl acetate (1:1;100 ml). The organic phase wasevaporated and the residue purified by flash chromatography usinghexane:ethyl acetate (2:1) as eluent to give the title compound (124mg).

δ (¹ H, CDCl₃): 10.01 (s, 1H, CHO), 7.60 (m, 10H, 2×Ph), 7.26 (s, 1H,CH-Ph₂), 6.31 (dd, 1H, H-2, J=1.5 and 3.3 Hz), 4.87 (s, 1H, H-1', J=1.2Hz), 4.40 (t, 1H, H-3', J=3.3 Hz), 4.35 and 3.94 (d,d, 1H, 1H, 8a-CH₂,J=9 Hz), 4.04 (m, 2H, H-2' and H-5'), 3.80 (m, 2H, OCH₂ CH₂), 3.44 (dd,1H, H-4', J=3.3 and 9 Hz), 3.01 (t, 1H, H-1, J=3.6 Hz).

INTERMEDIATE 4

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-2,3,4-tri-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of Intermediate 1 (0.6 mmol) in dry tetrahydrofuran (20 ml)under nitrogen was treated with sodium hydride (1.5 mmol). After 30minutes, methyl iodide (112 μl) was added. The mixture was stirred atroom temperature for 4 hours and poured into ethyl acetate:water(1:1;100 ml). The organic phase was evaporated and the residue purifiedby flash chromatography using hexane:ethyl acetate (3:1) as eluent.Appropriate fractions were concentrated to give the title compound (195mg).

δ (¹ H, CDCl₃): 9.77 (s, 1H, CHO), 7.37 (m, 10H, 2×Ph), 6.99 (s, 1H,CHPh₂), 6.05 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 4.55 (d, 1H, H-1', J=0.9Hz), 4.07 and 3.69 (d,d, 1H, 1H, 8a-CH₂, J=8.7 Hz), 3.71 (m, 2H, H-2'and H-3'), 3.43 (dd, 1H, H-5', J=0.6 and 3.9 Hz), 3.57 (s, 3H,2'--OCH₃), 3.51 (s, 3H, 3'--OCH₃), 3.16 (dd, 1H, H4', J=3 and 9.3 Hz),2.75 (t,1H, H-1, J=3.9 Hz).

INTERMEDIATE 5

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of 4'-demethylsordarin (10 g) in methanol (20 ml) wasadded dropwise at room temperature a solution of diphenyidiazomethane(90 ml) in methylene chloride, and the mixture was stirred for 6 hours.The solvent was evaporated to dryness and the residue chromatographed ina silica gel flash column with n-hexane:ethyl acetate (3:1) as eluent togive the title compound (12.6 g) as a pale yellow foam.

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 6.98 (s, 1H, CHPh₂), 6.05 (dd, 1H,H-2, J=1.5 and 3.6 Hz), 4.65 (d, 1H, H-1', J=1.5 Hz), 4.09, 3.76 (2d,2H, 8a-CH₂, J=9 Hz), 4.01 (m, 1H, H-2'), 3.84 (m, 1H, H-3'), 3.75 (m,1H, H-5'), 3.69 (m, 1H, H-4'), 2.73 (t, 1H, H-1, J-4.2 Hz).

INTERMEDIATE 6

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-3,4-O-isopropylidene-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-(1H)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 5 (650 mg) in 15 ml of2,2-dimethoxypropane:acetone (1:2) was added p-toluensulphonic acid (10mg). The solution was stirred at room temperature for 1.5 hours, thenpotassium carbonate (1.0 g) was added, the stirring continued for 30minutes and the solvent evaporated to dryness. The crude mixture waspartitioned between ethyl acetate (50 ml) and water (25 ml), the aqueousphase was extracted with ethyl acetate (2×50 ml), the organic phase waswashed with brine, dried over magnesium sulphate and evaporated todryness. The residue was flash chromatographed on silica gel elutingwith ethyl acetate:hexane (1:3) to give the title compound (600 mg) as awhite foam.

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 7.45-7.24 (m, 10H, 2Ph), 6.98 (s, 1H,CHPh₂), 6.06 (dd, 1H, H-2, J=1.5 and 3.3 Hz), 4.57 (d, 1H, H-1', J=3.0Hz), 4.30 (dd, 1H, H-3', J=3.6 and 5.7 Hz), 4.07 (d, 1H, 8aCH₂, J=9.0Hz), 3.95-3.93 (m, 1H, H-2'), 3.85 (dd, 1H, H4', J=5.7 and 9.3 Hz), 3.75(d, 1H, 8aCH₂, J=9.0 Hz), 3.44 (dq, 1H, H-5', J_(d) =9.3 Hz, J_(q) =6.3Hz), 2.73 (t, 1H, H-1, J=3.6 Hz).

INTERMEDIATE 7

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-3,4-O-isopropylidene-2-O-(methylthio)thiocarbonyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

Intermediate 6 (100 mg) and imidazole (1 mg) were dissolved in drytetrahydrofuran (4 ml) under nitrogen atmosphere. Sodium hydride (5 mg)was added and the suspension was stirred at room temperature for 0.5hours Carbon disulfide (2.7 ml) was added, the stirring continued for 20minutes and methyl iodide (18 ml) was added. After 2 hours the reactionwas stopped by addition of 1N ammonium chloride (20 ml). The mixture wasextracted with ethyl acetate (3×25 ml), the organic phase was washedwith brine, dried over magnesium sulphate and the solvent evaporated todryness. The residue was purified in a flash chromatography column onsilica gel eluting with ethyl acetate:hexane (1:9) to give the titlecompound (110 mg).

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 7.44-7.25 (m, 10H, 2Ph), 6.96 (s, 1H,CHPh₂), 6.01 (dd, 1H, H-2, J=1.2 and 3.6 Hz), 6.90 (dd, 1H, H-2', J-2.4and 5.4 Hz), 4.85 (d, 1H, H-1', J=2.4 Hz), 4.53 (dd, 1H, H-3', J=5.4 and6.3 Hz), 4.00 (dd, 1H, H-4', J=6.3 and 8.7 Hz), 3.93 (d, 1H, 8aCH₂,J=9.3 Hz), 3.65 (dq, 1H, H-5', J_(d) =8.7, J_(q) =6.3 Hz), 2.68 (t, ₁ H,H-1, J=3.9 Hz), 2.59 (s, 3H, CH₃ S).

INTERMEDIATE 8

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,6-Dideoxy-3,4-O-isopropylidene-β-D-allopyranosyloxy)methyl]-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

Intermediate 7 (95 mg) was dissolved in dry toluene (5 ml) undernitrogen atmosphere and heated at 110° C. A solution of tributyltinhydride (64 ml) in dry toluene (5 ml) was added dropwise over 1.5 hourswith stirring. The heating was continued for another 1.5 hours, methanol(2 ml) was added and the solvent evaporated to dryness. Flashchromatography of the residue on silica gel eluting with ethylacetate:hexane (1:9) gave the title compound (42 mg).

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 7.44-7.25 (m, 10H, 2Ph), 6.98 (1H, s,CHPh₂), 6.05 (dd, 1H, H-2, J=1.2 and 3.3 Hz), 4.54 (dd, 1H, H-1', J=2.7and 9.3 Hz), 4.39 (dt, 1H, H-3', J_(d) =2.7 Hz, J_(t) =3.6 Hz), 4.04 (d,1H, 8aCH₂, J=9.0 Hz),3.67 (d, 1H, 8aCH₂, J=9.0 Hz), 3.65 (dd, 1H, H4',J=3.6 and 8.7 Hz), 3.44 (dq, 1H, H-5', J_(d) =6.3 Hz, J_(q) =8.7 Hz),2.75 (t, 1H, J=3.9 Hz).

INTERMEDIATE 9

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,6-Dideoxy-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 8 (1.5 g) in a mixture of tetrahydrofuran(30 ml) and methanol (15 ml) was added dropwise at room temperature a 1Nsolution of hydrochloric acid (15 ml) with vigorous stirring. Once thereaction was concluded (TLC control), saturated sodium bicarbonate (50ml) and ethyl acetate (200 ml) were added and the mixture partitioned.The organic layer was washed with water (2×100 ml) and dried overmagnesium sulfate. Elimination of the solvent gave a residue which wasflash chromatographed over silica gel eluting with hexane:ethyl acetate(5:1) and (2:1) to give the title compound (1.1 g) as a white foam.

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 7.5-7.2 (m, 10H, 2Ph), 6.99 (s, 1H,CHPh₂), 6.05 (dd, 1H, H-2, J=1.2 and 3.3 Hz), 4.64 (dd, 1H, H-1', J=2.1and 9.6 Hz), 4.11 (m, 1H, H-3'), 4.06 (d, 1H, 8aCH₂, J_(AB) =9.3 Hz),3.70 (m, 2H, H-5' and 8aCH₂), 3.34 (m, 1H, H-4'), 2.75 (t, 1H, H-1,J=3.6 Hz).

INTERMEDIATE 10

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,6-Dideoxy-4-O-methyl-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A suspension of Intermediate 9 (380 mg) and dibutyltin oxide (225 mg) indry toluene (15 ml) was refluxed for 2 hours in a flask fitted with aDean Stark condenser, under nitrogen. The mixture was allowed to standat room temperature and methyl iodide (200 μl) and a 1M solution oftetrabutylammonium fluoride in tetrahydrofuran (3 ml, 3 mmol) were addedconsecutively in several portions. The mixture was heated at 40° C. for24 hours, under nitrogen. The solvent was then evaporated in vacuo andthe residue purified by silica gel flash chromatography eluting withhexane:acetone (10:1) to afford the title compound (220 mg) togetherwith the corresponding 3-O-methyl compound which eluted with a lower Rf.

δ (¹ H, CDCl₃): 9.72 (s, 1H, CHO), 7.32 (m, 10H, 2Ph), 6.98 (s, 1H,CHPh2), 6.05 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 4.64 (dd, 1H, H-1', J-1.8and 9.6 Hz), 4.25 (q, 1H, H-3', J=3.3 Hz), 4.52 and 3.67 (2d, 2H,8a-CH₂, J=9.3 Hz), 3.69 (m, 1H, H-5'), 3.41 (s, 3H, OCH₃), 2.87 (dd, 1H,H4', J=3.3 and 9.3 Hz), 2.75 (t, 1H, H-1).

INTERMEDIATE 11

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,6-Dideoxy-4-O-methyl-3-O-methyloxymethyl-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 10 (200 mg) in diisopropylethylamine (4ml) at 0° C. under nitrogen was added a large excess of methyloxymethylchloride (0.2 to 1 ml). The mixture was stirred for 1 hour at 0° C. and24 hours at room temperature. The reaction was quenched with theaddition of methanol, 10% sodium bicarbonate and hydrochloric acid, andstirring was continued for 2 hours. Water (50 ml) and ethyl acetate (50ml) were added and the organic layer was separated and dried overmagnesium sulphate. Removal of the solvent gave the title compound (200mg).

δ (¹ H, CDCO₃): 9.74 (s, 1H, CHO), 6.98 (s, 1H, CHPh₂), 6.04 (dd, 1H,H-2, J=1.5 and 3.6 Hz), 4.74 (AB system, 2H, OCH₂ OCH₃, J=6.6 Hz), 4.61(dd, 1H, H-1', J=2.1 and 9.3 Hz), 4.20 (m, 1H, H-3'), 4.04 and 3.69 (2d,2H, 8a-CH₂, J=9.6 Hz), 3.81 (m, 1H, H-5'), 3.41 (s, 3H, OCH₃), 3.40 (s,3H, OCH₃), 2.88 (dd, 1H, H-4', J=3 and 9.6 Hz), 2.75 (t, 1H, H-11, J=3.9Hz).

INTERMEDIATE 12

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-(1,3-dioxolan-2-yl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of Intermediate 1 (9.7 mmol) in dry methanol (40 ml) wastreated with ethylene glycol (110 ml), trimethylorthoformate (3.25 ml)and a catalytic amount of p-toluenesulfonic acid. The mixture was heatedat 40° C. for 3 hours. After cooling, the mixture was poured into ethylacetate:aqueous sodium hydrogen carbonate (1:1; 500 ml) and the waterlayer was thoroughly extracted with ethyl acetate. The combined organiclayers were washed with water and brine, and dried. After removal of thesolvent, the residue was purified by flash chromatography usinghexane:ethyl acetate (6:1) as eluent to give the title compound (6.1 g).

δ (¹ H, CDCl₃): 7.43 and 7.30 (m,m, 4H, 6H, 2×Ph), 6.94 (s, 1H, CHPh₂),5.83 (dd, 1H, H-2, J=1.2 and 3.6 Hz), 5.07 (s, 1H, 4-CH), 4.63 (d, 1H,H-1', J=1.5 Hz), 4.20 (t, 1H, H-3', J=3.3 Hz), 4.07 (d, 1H, 8a-CHa,J=9.3 Hz), 3.84 (m, 6H, H-2', 8a-CHb and OCH₂ CH₂ O), 3.71 (m, 1H,H-5'), 3.42 (s, 3H, OCH₃), 3.21 (dd, 1H, H4', J=3 and 9 Hz), 2.63 (m,1H, CH(CH₃)₂), 2.54 (t, 1H, H-1, J=3.9 Hz).

INTERMEDIATE 13

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[[6-Deoxy-4-O-methyl-2,3-di-O-(p-tolylsulfonyl)-β-D-altropyranosyloxy]methyl]-4-(1,3-dioxolan-2-yl)-4,4a,5,6,7,7a8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 12 (3.88 mmol) and 4-dimethylaminopyridine(9 mmol) in dry dichloromethane (15 ml) was added dropwise a solution ofp-toluenesulfonyl chloride (8 mmol) in dry dichloromethane (15 ml).After stirring for one day, the mixture was washed with water and theorganic layer was evaporated. The residue was purified by flashchromatography using hexane:ethyl acetate (4:1) as eluent. Appropriatefractions were combined and evaporated to dryness to give the titlecompound (3.1 g).

δ (¹ H, CDCl₃): 7.88 and 7.30 (m,m, 4H, 14H, Ar), 6.90 (s, 1H, CHPh₂),5.64 (dd, 1H, H-2, J=1.2 and 3.6 Hz), 5.01 (s, 1H, 4-CH), 4.98 (dd, 1H,H-3', J=3 and 4.2 Hz), 4.59 (m, 2H, H-1' and H-2'), 3.94 and 3.53 (d,d,1H, 1H, 8a-CH₂, J=9 Hz), 3.82 (m, 4H, OCH₂ CH₂ O), 3.68 (m, 1H, H-5'),3.18 (dd, 1H, H4', J=3 and 9 Hz), 2.83 (s, 3H, OCH₃), 2.55 (m, 1H,CH(CH₃)₂), 2.48 and 2.45 (s,s, 3H, 3H, 2×CH₃ Ar).

INTERMEDIATE 14

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,3-Anhydro-6-deoxy-4-O-methyl-β-D-allopyranosyloxy)methyl]-4-(1,3-dioxolan-2-yl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of Intermediate 13 (2.87 mmol) in dry dichloromethane (20ml), under nitrogen atmosphere at 0° C., was treated with a solution ofsodium methoxide (20 mmol) in methanol (20 ml). After three days thesolvent was evaporated to dryness and the residue was dissolved indichloromethane:water (1:1; 100 ml). The organic layer was evaporatedand the residue purified by flash chromatography using hexane:ethylacetate (7:1) as eluent. Appropriate fractions eluting with the higherRf were combined and evaporated to give the title compound (680 mg).

δ (¹ H, CDCl₃): 7.44 and 7.29 (m,m, 4H, 6H, 2×Ph), 6.93 (s, 1H, CHPh₂),5.86 (dd, 1H, H-2, J=0.9 and 3.3 Hz), 5.08 (s, 1H, 4-CH), 4.59 (s, 1H,H-1'), 4.03 (d, 1H, 8a-CHa, J=9 Hz), 3.83 (m, 5H, 8a-CHb and OCH₂ CH₂O), 3.49 (m, 4H, H4' and OCH₃), 3.33 (m, 3H, H-2', H-3' and H-5'), 2.64(m, 1H, CH(CH₃)₂), 2.58 (t, 1H, H-1, J=3.9 Hz).

INTERMEDIATE 15

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,3-Anhydro-6-deoxy-4-O-methyl-β-D-mannopyranosyloxy)methyl]-4-(1,3-dioxolan-2-yl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid diphenylmethyl ester

Appropriate fraction from the Intermediate 14 preparation eluting at thelower, Rf were combined and evaporated to give the title compound (912mg).

δ (¹ H, CDCl₃): 7.43 and 7.29 (m, m, 4H, 6H, 2×Ph), 6.95 (s, 1H, CHPh₂),5.86 (dd, 1H, H-2, J=1.2 and 3.6 Hz), 5.07 (s, 1H, 4-CH),4.66 (s, 1H,H-1'), 4.11 (d, 1H, 8a-CHa, J=9.3 Hz), 3.82 (m, 5H, 8a-CHb and OCH₂ CH₂O), 3.49 (s, 3H, OCH₃), 3.24 (d, 1H, H-2', J=3.9 Hz), 3.16 (m, 1H,H-5'), 3.12 (m, 2H, H-3' and H-4'), 2.62 (m, 2H, H-1 and CH(CH₃)₂).

INTERMEDIATE 16

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-Azido-3,6-dideoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-(1,3-dioxolan-2-yl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of Intermediate 15 (0.7 mmol) and lithium azide (2.9 mmol) indry dimethylformamide (10 ml) was heated for 45 hours at 100° C. Aftercooling, the mixture was poured into ethyl acetate:water (1:1;100 ml)and the organic phase was evaporated. The residue was purified by flashchromatography using hexane:ethyl acetate (4:1) as eluent. Appropriatefractions were combined and concentrated to give the title compound (359mg).

(¹ H, CDCl₃): 7.43 and 7.30 (m,m, 4H, 6H, 2×Ph),6.93 (s, 1H, CHPh₂),5.83 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 5.07 (s, 1H, 4-CH), 4.50 (d, 1H,H-1', J=1.5 Hz), 4.14 (m, 1H, H-2'), 4.03 (d, 1H, 8a-CHa), 3.79 (m, 7H,H-3', H-5', 8a-CHb and OCH₂ CH₂ O), 3.45 (s, 3H, OCH₃), 3.36 (dd, 1H,H4', J=3.3 and 8.1 Hz), 2.64 (m, 1H, CH(CH₃)₂), 2.49 (t, 1H, H-1, J=4.2Hz), 2.36 (d, 1H, 2'--OH, J=3.3 Hz).

INTERMEDIATE 17

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3,6-Dideoxy-4-O-methyl-3-methylthio-β-D-altropyranosyloxy)methyl]-4-(1,3-dioxolan-2-yl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A mixture of Intermediate 15 (0.58 mmol) and sodium thiomethoxide (3mmol) in dry dimethylformamide (5 ml) was stirred for 48 hours and thenpoured into ethyl acetate:water (1:1;100 ml). The organic phase wasevaporated and the residue purified by flash chromatography usinghexane:ethyl acetate (4:1) as eluent. Appropriate fractions werecombined and evaporated to give a mixture which was dissolved in drydichloromethane (10 ml) and treated with 4-dimethylaminopyridine (0.7mmol). After stirring for 20 minutes at -20° C. under nitrogenatmosphere, trichloroethoxycarbonyl chloride (0.35 mmol) was added.After 90 minutes, the mixture was washed with water, the organic phasewas evaporated and the residue was purified by flash chromatographyusing hexane:ethyl acetate (4:1) as eluent to give a solid which wasdissolved in tetrahydrofuran (10 ml) and treated with 1M aqueouspotassium phosphate (0.3 ml) and zinc (4.6 mmol). The mixture wasstirred overnight at room temperature, filtrated and washed with water.The organic phase was evaporated and the residue purified by flashchromatography using hexane:ethyl acetate (5:1) as eluent to give thetitle compound (40 mg).

δ (¹ H, CDCl₃): 7.44 and 7.30 (m, m, 4H and 6H, 2×Ph), 6.94 (s, 1H,CHPh₂), 5.85 (dd, 1H, H-2, J=0.9 and 3.3 Hz), 5.07 (s, 1H, 4-CH), 4.69(d, 1H, H-1', J=1.5 Hz), 4.04 (d, 1H, 8a-CHa, J=9Hz), 3.94 (m, 1H,H-2'), 3.82 (m, 5H, 8a-CHb and OCH₂ CH₂ O), 3.73 (m, 1H, H-5'), 3.47(dd, 1H, H-4', J=4.2 and 8.4 Hz), 3.40 (s, 3H, OCH₃), 3.33 (t, 1H, H-3',J=4.2 Hz), 2.63 (m, 1H, CH(CH₃)₂), 2.52 (t,1 H, H-1, J=4.2 Hz), 2.44(d,1H, 2'--OH, J=3.6 Hz), 2.23 (s, 3H, SCH₃).

INTERMEDIATE 18

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-Azido-3,6-dideoxy-4-O-methyl-β-D-glucopyranosyloxy)methyl]-4-(1,3-dioxolan-2-yl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of Intermediate 14 (0.2 mmol) and lithium azide (0.7 mmol) indry dimethylformamide (5 ml) was heated for 48 hours at 100° C. Aftercooling, the mixture was poured into ethyl acetate:water. (1:1;50 ml).The organic phase was evaporated and the residue purified by flashchromatography using hexane:ethyl acetate (3:1) as eluent. Appropriatefractions were combined and concentrated to give the title compound (71mg).

δ (¹ H, CDCl₃): 7.31 (m, 10H, 2×Ph), 6.92 (s, 1H, CHPh₂), 5.84 (dd, 1H,H-2, J=1.2 and 3.3 Hz), 5.08 (s, 1H, 4-CH), 4.04 (m, 2H, H-1' and8a-CHa), 3.84 (m, 5H, 8a-CHb and OCH₂ CH₂ O), 3.32 (m, 3H, H-2', H-3'and H-5'), 2.74 (t, 1H, H-4', J=9 Hz), 2.66 (m, 1H, CH(CH₃)₂), 2.54 (t1H, H-1, J=3.6 Hz), 2.30 (d, 1H, 2'--OH, J=1.8 Hz).

INTERMEDIATE 19

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3,6-Dideoxy-4-O-methyl-3-methylthio-β-D-glucopyranosyloxy)methyl]-4-(1,3-dioxolan-2-yl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of Intermediate 14 (0.3 mmol) and sodium thiomethoxide (1mmol) in dry dimethylformamide (5 ml) was stirred for 26 hours and thenpoured into ethyl acetate:water (1:1;100 ml). The organic phase wasevaporated and the residue purified by flash chromatography usinghexane:ethyl acetate (4:1) as eluent to give the title compound (134mg).

δ (¹ H, CDCl₃): 7,33 (m,10H, 2×Ph), 6.93 (s, 1H, CHPh₂), 5.86 (dd, 1H,H-2, J=1.2 and 3.6 Hz), 5.08 (s, 1H, 4-CH), 4.10 (m, 2H, H-1', and8a-CHa), 3.84 (m, 5H, 8a-CHb and OCH₂ CH₂ O), 3.60 (s, 3H, OCH₃), 3.30(m, 3H, H-2', H-3' and H-5'), 2.80 (m, 1H, H-4'), 2.65 (m, 2H, H-1 andCH(CH₃)₂), 2.21 (s, 3H, SCH₃).

INTERMEDIATE 20

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2-O-Acetyl-3-acetylamino-3,6-dideoxy-4-O-methyl-β-D-glucopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a mixture of Intermediate 18 (0.4 mmol) in methanol (40 ml) and 1Nhydrochloric acid (10 ml) was added 10% palladium on charcoal (70 mg)under nitrogen. The mixture was shaken in a Parr apparatus under 20 psiof hydrogen for 2 hours at room temperature. The catalyst was filteredoff and the solvent evaporated to dryness. The residue was dissolved indichloromethane (5 ml) and treated with pyridine (3 ml) and aceticanhydride (0.6 mmol). The mixture was heated for 48 hours at 40° C. andthen cooled and washed with water. The organic phase was evaporated andthe residue purified by flash chromatography usingdichloromethane:methanol (20:1) as eluent to give the title compound(167 mg).

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 6.00 (dd, 1H, H-2, J=1.2 and 3.3 Hz),5.55 (d, 1H, NH, J=9.3 Hz), 4.66 (dd, 1H, H-2', J=7.8 and 10.5 Hz), 4.28(m, 2H, H-1' and H-3'), 4.06 and 3.54 (d,d, 1H, 1H, 8a-CH₂, J=9.6 Hz),3.49 (m, 4H, OCH₃ and H-5'), 2.82 (t, 1H, H4', J=9.6 Hz), 2.58 (t, 1H,H-1, J=4.2 Hz), 2.32 (m, 1H, CH(CH₃)₂), 2.01 and 1.99 (s,s, 3H, 3H,2×CH₃ CO).

INTERMEDIATE 21

[1R(1α, 3aβ, 4β, 4aβ, 7β, 7aβ, 8aβ)]8a-[(2,3,6-Trideoxy-3-iodo-β-D-glucopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

Intermediate 9 (2.0 g), triphenylphosphine (3.4 g) and imidazole (186mg) were refluxed in toluene (50 ml) with stirring and then treated withiodine (610 mg), added in small portions. Refluxing was continued for 4hours. The reaction mixture was cooled, decanted into excess aqueoussodium hydrogen carbonate and sodium thiosulfate in a separating funnel.The mixture was shaken until the iodine was consumed. The toluene phasewas washed with water, dried over magnesium sulfate and concentrated.The residue was purified by flash column chromatography eluting withhexane:ethyl acetate (15:1) and (5:1) to give the title compound (1.65g).

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 7.26-7.44 (m, 10H, 2Ph), 6.98 (s, 1H,CHPh₂), 6.04 (dd, 1H, H-2, J-1.2 and 3.3 Hz), 4.25 (dd, 1H, H-1', J=1.8and 9.3 Hz), 4.08 (m, 1H, H-4'), 3.67 and 4.01 (2d, 2H, 8aCH₂, J=9 Hz),3.40 (m, 1 H, H-3'), 3.32 (m 1H, H-5'), 2.74 (t, 1H, H-1, J=4.2 Hz),2.32 and 2.53 (2m, 2H, 2H-240 ).

INTERMEDIATE 22

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα,8aβ)]-[(3-Azido-2,3,6-trideoxy-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A stirred solution of Intermediate 21 (300 mg) in dry dimethylformamidewas treated with lithium azide (60 mg). The reaction mixture was heatedat 70° C. for 18 hours. The solvent was removed under reduce pressureand the residue was purified by flash column chromatography eluting withhexane:ethyl acetate (4:1) to afford the title compound (246 mg) as apale yellow syrup.

δ (¹ H, CDCl₃): 9.74 (s, 1H, CHO), 7.25-7.45 (m, 10H, 2Ph₂), 6.97 (s,1H, CHPh₂), 6.07 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 4.52 (dd, 1H, H-1',J=1.4 and 9.3 Hz), 4.07 (q, 1H, H-3', J=3.6 Hz), 3.68 and 4.02 (2d, 2H,8aCH₂, J=9.3 Hz), 3.59 (dq, 1H, H-5', J=6.3 and 9 Hz), 3.40 (dd, 1H,H4', J=3.6 and 9 Hz), 2.74 (t, 1H, H-1, J=3.6 Hz), 2.06-2.12 and1.73-4.82 (2m, 2H, 2H-2').

INTERMEDIATE 23

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-Azido-4-Azido-4-O-methyl-2,3,6-trideoxy-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a stirred solution of Intermediate 22 (140 mg) in dry tetrahydrofuran(10 ml) at 0° C. was added sodium hydride (97%, 6 mg). The mixture wasstirred for 30 minutes under nitrogen, before methyl iodide (0.7 ml) wasadded. Stirring was continued for a further 5 hours at which time thereaction was quenched with methanol (3 ml). The solvent was evaporatedto dryness under reduce pressure and the residue was purified by flashcolumn chromatography eluting with hexane:ethyl acetate (6:1) to affordthe title compound (56 mg).

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 7.44-7.26 (m, 10H, 2Ph), 6.98 (s, 1H,CHPh₂), 6.06 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 4.50 (dd, 1H, H-1', J=2.1and 9.3 Hz), 4.22 (m, 1H, H-3'), 3.67 and 4.03 (2d, 2H, 8aCH₂, J=9.3Hz), 3.76 (dq, 1H, H-5', J=6 Hz and 9 Hz), 3.44 (s, 3H, 4'--OMe), 3.01(dd,1H, H-4', J=3.3 and 9 Hz).

INTERMEDIATE 24

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2-O-Benzyloxycarbonyl-6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 1 (3 mmol) in dry dichloromethane (15 ml)at 0° C. under nitrogen atmosphere was added 4-dimethylaminopyridine(6.3 mmol). After stirring for 15 minutes, the mixture was cooled to-20° C. and a solution of benzylchloroformate (3.6 mmol) in drydichloromethane (15 ml) was added dropwise. The solvent was evaporatedand the residue purified by flash chromatography using hexane:ethylacetate (3:1) as eluent to give the title compound (1.2 g).

δ (¹ H, CDCl₃): 9.70 (s, 1H, CHO), 7.35 (m, 15H, 3×Ph), 6.96 (s, 1H,CHPh₂), 5.99 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 5.18 (AB system, 2H,OCH2Ph, J=12 Hz), 5.00 (dd, 1H, H-2', J=1.5 and 4.2 Hz), 4.70 (d, 1H,H-1', J=1.8 Hz), 4.13 (m, 1H, H-3'), 4.01 and 3.66 (d,d, 1H, 1H, 8a-CH₂,J=9 Hz), 3.75 (m, 1H, H-5'), 3.40 (s, 3H, OMe), 3.17 (dd, 1H, H4', J=3.3and 8.4 Hz), 2.62 (t, 1H, H-1, J=3.6 Hz), 2.43 (d, 1H, OH, J=2.4 Hz),2.21 (m, 1H, CH(CH₃)₂).

INTERMEDIATE 25

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2-O-Benzyloxycarbonyl-6-deoxy-4-O-methyl-3-O-(2-tetrahydropyranyl)-.beta.-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A mixture of Intermediate 24 (0.300 g) and 2,3-dihydropyran (0.138 ml)in dichloromethane (10 ml) containing pyridinium p-toluenesulphonate(0.019 g) was stirred at room temperature for 20 hours. After removal ofthe solvent in vacuo, the residue was chromatographed on silica gelflash column eluting with 1% dichloromethane in methanol to afford thetitle compound (0.208 g) as a 3:2 diastereomeric mixture.

δ (¹ H, CDCl₃) only major diastereomer: 9.72 (s, 1H, CHO), 7.43 to 7.25(m, 15H, 3'Ph), 6.97 (s, 1H, Ph₂ CH), 6.00 (dd, 1H, H-2, J=1.2 and 3.0Hz), 5.16 (sist AB, 2H, PhCH₂ OCO), 5.04 (dd, 1H, H-2', J=1.2 and 4.2Hz), 4.80 (m, 1H, H-1"), 4.68 (brs, 1H, H-1'), 4.21 (dd, 1H, H-3', J=3.0and 4.5 Hz).

INTERMEDIATE 26

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,3,6-Trideoxy-2,3-difluoro-4-O-methyl-β-D-glucopyranosyloxy)methyl]-4-(1,3-dioxolan-2-yl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of Intermediate 12 (500 mg) in anhydrous dichloromethane (8ml) at -40° C. was treated with diethylaminosulfur trifluoride (DAST,0.35 ml). The cooling bath was removed and the mixture was stirredovernight at room temperature. The mixture was cooled to -20° C., thereaction quenched by addition of methanol (2 ml) and then concentratedunder reduced pressure. Flash chromatography on silica gel eluting withhexane:ethyl acetate (20:1), (10:1) and (4:1) gave the title compound(140 mg) as a white foam.

δ (¹ H, CDCl₃): 7.26-7.46 (m, 10H, 2Ph), 6.89 (s, 1H, CHPh₂), 5.87 (dd,1H, H-2, J=1.2 and 3.6 Hz), 5.07 (s, 1H, CH-dioxolane), 4.58-4.70 and4.41-4.52 (2m, 1H, H-2', J_(HF) =53.1 and 17.1, J_(HH) =8.7 and 8.1 Hz),4.23 (d, 1H, H-1', J=8.4 Hz), 4.30-4.40 and 4.12-4.21 (2m, 1H, H-3',J_(HF) =53.1 and 15.3 Hz, J_(HH) =8.1 Hz), 4.05 (d, 1H, 8aCH₂, J=9.3Hz), 3.77-3.89 (m, 4H, of 2CH₂ in dioxolane protecting group and 1H of8aCH₂), 3.55 (d, 3H, 4'--OCH₃, J_(HF) =1.2 Hz), 3.20-3.30 (m, 1H, H-5'),2.96-3.07 (dq, 1H, H4', J_(HF) =13.2 Hz, J_(HH) =8.4 and 9.6 Hz),2.60-2.70 (m, 2H, H-1 and 2CH isopropyl).

INTERMEDIATE 27

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,3,6-Trideoxy-4-O-methyl-3-oxo-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of trifluoroacetic anhydride (0.1 ml) in dry dichloromethane(5 ml) at -60° C. under nitrogen was treated with dimethylsulfoxide(0.06 ml). After 10 minutes, a solution of Intermediate 10 (0.39 mmol)in dry dichloromethane (5 ml) was added followed, after 60 minutes, bytriethylamine (0.24 ml). The mixture was stirred at -60° C. to -20° C.for 2 hours and washed with water. After removal of the solvent, theresidue was purified by flash chromatography using hexane:ethyl acetate(3:1) as eluent to give the title compound (131 mg).

δ (¹ H, CDCl₃): 9.72 (s, 1H, CHO), 7.35 (m, 10H, 2×Ph), 6.97 (s, 1H,CHPh₂), 6.03 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 4.46 (dd, 1H, H-1', J=2.7and 8.9 Hz), 4.07 and 3.71 (d,d, 1H, 1H, 8a-CH₂, J=9 Hz), 3.42 (m, 2H,H4' and H-5'), 2.70 (m, 3H, H-1 and CH₂ --2'), 2.25 (m, 1H, CH(CH₃)₂),1.43 (d, 3H, CH₃ --6', J=5.7 Hz).

INTERMEDIATE 28

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3,6-Dideoxy-2-O-benzyloxycarbonyl-4-O-methyl-3-oxo-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of trifluoroacetic anhydride (65 μl) in drydichloromethane (2 ml) under nitrogen atmosphere at -60° C. were addeddropwise dimethylsulphoxide (33 μl) and a solution of Intermediate 24(250 mg) in dichloromethane. The resulting solution was stirred for 40minutes, triethylamine (0.20 ml) was added and the reaction mixture keptat -20° C. overnight. The mixture was then poured into water, theaqueous phase extracted with dichloromethane, the organic phase washedwith brine, dried (magnesium sulphate) and evaporated to dryness. Flashchromatography on silica gel eluting with ethyl acetate:hexane (2:8)afforded the title compound (152 mg) as a foam.

δ (¹ H, CDCl₃): 9.69 (s, 1H, CHO), 7.43-7.25 (m, 10H, 2Ph), 6.95 (s, 1H,CHPh₂), 5.91 (dd, 1H, H-2, J=1.5 and 3.3 Hz), 5.27 (d, 1H, PhCH₂ O,J=12.0 mHz), 5.20 (d, 1H, PhCH₂ O, J=12.0 Hz), 4.99 (dd, 1H, H-2', J=1.2and 7.8 Hz), 4.37 (d, 1H, H-1', 7.8 Hz), 4.07 (d, 1H, 8aCH₂, J=9.0 Hz),3.71 (d, 1H, 8aCH₂, J=12.0 Hz), 3.53 (s, 3H, 4'--O--CH₃), 3.51 (dd, 1H,H-4', J=1.2 and 9.6 Hz), 3.42 (dq, 1H, H-5', J_(d) =9.6 Hz, J_(q) =6.0Hz), 2.62 (t, 1H, H-1, J=3.9 Hz).

INTERMEDIATE 29

[1R(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-2-O-benzyloxycarbonyl-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-(1,3-dioxolan-2-yl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 24 (2.03 g) in methanol (18 ml) were addedethylene glycol (30 ml), methyl orthoformate (2.5 ml) andp-toluensulphonic acid (31 mg), and the resulting solution was stirredunder nitrogen atmosphere at 35° C. for 3 hours. The solution was thenpartitioned between 10% sodium bicarbonate and ethyl acetate (100 ml),and the aqueous phase was extracted with more ethyl acetate (2×100 ml),washed with brine, dried (magnesium sulphate) and evaporated to dryness.The residue was flash chromatographed on silica gel eluting withhexane:ethyl acetate (6:4) to yield the title compound (2.3 g).

δ (¹ H, CDCl₃): 7.46-7.20 (m, 10H, 2Ph), 6.92 (s, 1H, CHPh₂),5.97 (dd,1H, H-2, J=1.2 and 3.6 Hz), 5.22 (d, 1H, BnCH₂ O, J=12.3 Hz), 5.14 (d,1H, BnCH₂ O, J=12.3 Hz), 5.05 (s, 1H, OCHO), 4.99 (dd, 1H, H-2', J=1.8and 4.5 Hz), 4.69 (d, 1H, H-1', J=1.8 Hz), 4.19-4.15 (m, 1H, H-3'), 4.01(d, 1H, 8aCH₂, J=9.0 Hz), 3.89-3.74 (m, 5H, H-5', 2CH₂ O), 3.71 (d, 1H,8aCH₂, J=9.0 Hz), 3.40 (s, 3H, 4--O--CH₃), 3.17 (dd, 1H, H4', J=3.3 and8.4 Hz).

INTERMEDIATE 30

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3,6-Dideoxy-2-O-benzyloxycarbonyl-4-O-methyl-3-oxo-β-D-allopyranosyloxy)methyl]-4-(dioxolan-2-yl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of trifluoroacetic anhydride (0.64 ml) in drydichloromethane (5 ml) under nitrogen atmosphere at -60° C. were addeddropwise dimethylsulphoxide (0.35 ml) and a solution of Intermediate 29(1.9 g) in dry dichloromethane (10 ml). The resulting solution wasstirred for 1 hour, triethylamine (1.24 ml) was added and stirring wascontinued for 2 hours allowing the temperature to reach -20° C. Thereaction mixture was partitioned between water and dichloromethane (100ml), and the organic phase was washed with brine and dried (magnesiumsulphate). The solution was then concentrated to 40 ml, triethylamine (2ml) was added and the solution stirred for 2 hours at room temperature.The solvent was then evaporated off and the residue flashchromatographed on silica gel eluting with ethyl acetate:hexane (2:8) toobtain the title compound (1.6 g).

δ (¹ H, CDCl₃): 7.45-7.22 (m, 10H, CHPh₂), 5.73 (dd, 1H, H-2, J=1.5 and3.9 Hz), 5.28-5.19 (m, 2H, AB system, OCH₂ Ph), 5.08 (s, 1H, OCHO), 4.99(dd, 1H, H-2', J=1.2 and 8.1 Hz), 4.37 (d, 1H, H-1', J=8.1 Hz), 4.06 (d,1H, 8aCH₂, J=9.0 Hz), 3.88-3.78 (m, 4H, 2×CH₂ O), 3.52 (s, 3H,4--O--CH₃), 3.76 (d, 1H, BaCH₂, J=9.0 Hz), 3.46-3.39 (m, 1H, H-5').

INTERMEDIATE 31

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3,6-Dideoxy-2-O-benzyloxycarbonyl-3-allyloxyimine-4-O-methyl-β-D-allopyranosyloxy)methyl]-4-(1,3-dioxolan-2-yl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A mixture of Intermediate 30 (250 mg) and O-allylhydroxylaminehydrochloride (109 mg) in dry pyridine (5 ml) was stirred at 80° C. for4 hours. The solvent was evaporated to dryness and the residue flashchromatographed on silica gel eluting with hexane:ethyl acetate (85:15)to obtain the title compound (193 mg) as a mixture of Z:E isomers in a3:1 ratio.

δ (¹ H, CDCl₃): 7.43-7.21 (m, 10H, 2Ph), 6.91 (s, 1H, CHPh₂), 6.0-5.8(m, 1H, HC=C), 5.67 (d, 1H, H-2, J=6.6 Hz), 5.63 (dd, 1H, H-2, J=1.2 and2.4 Hz), 5.30-5.06 (m, 5H,2×H--C=C, OCHO, OCH₂ Ph), 4.74 (d, 1H, H-1',J=6.6 Hz), 4.68-4.58 (m, 2H, OCH₂ C=C), 4.04-3.75 (m, 6H, H-4', H-5'2×OCH₂), 3.97 (d, 1H, 8aCH₂, J=9.0 Hz), 3.66 (d, 1H, 8aCH₂, J=9.0 Hz),3.26 (s, 3H, 4--O--CH₃).

INTERMEDIATE 32

[1R-(1α, 3aβ, 4β, 4aβ7β, 7aα, 8aβ)]8a-[(3-O-Benzoyl-6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A stirred solution of Intermediate 1 (500 mg) and dibutyltin oxide (246mg) in dry toluene (15 ml) was refluxed for 2 hours in a flask fittedwith a Dean-Stark condenser under nitrogen atmosphere. The reactionmixture was allowed to cool to room temperature and then treated withbenzoyl chloride (95 μl) and triethylamine (0.32 ml). After stirring for2 hours under reflux, the reaction mixture was concentrated underreduced pressure and the residue purified by flash column chromatographyeluting with hexane:ethyl acetate (8:1 to 3:1). The appropriatefractions were combined and the solvents evaporated to yield the thetitle compound (300 mg) as a white foam.

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 8.05 and 7.26-7.59 (2m, 2H and 13H,3Ph), 6.99 (s, 1H, CHPh₂), 6.30 (dd, 1H, H-2, J=3.3 and 1.2 Hz), 5.73(dd, 1H, H-3', J=3 and 0.9 Hz), 4.69 (d, 1H, H-1', J=1.2 Hz), 3.79 and4.10 (2d, 2H, 8aCH₂, J=9 Hz), 3.94-3.85 (m, 2H, H-2' and H-5'), 3.38 (s,3H, 4'OMe), 3.41 (dd, 1H, H-4', J=3 and 9 Hz), 2.73 (t, 1H, H-1, J=3.9Hz), 2.38 (d, 1H, 2'--OH, J=3.3 Hz).

INTERMEDIATE 33

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-Benzyloxycarbonyl-6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid diphenylmethyl ester

A mixture of Intermediate 1 (3 mmol) and dibutyltin oxide (3.9 mmol) indry toluene (40 ml) was stirred under reflux in a flask fitted with aDean-Stark condenser for 2 hours. The mixture was cooled and4-dimethylaminopyridine (3.2 mmol) and benzyloxycarbonyl chloride (3.3mmol) were added. After 30 minutes, the solvent was evaporated and theresidue purified by flash column chromatography on silica gel elutingwith hexane:ethyl acetate (15:1 to 3:1). Appropriate fractions werecombined and concentrated to give the title compound (356 mg).

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 7.36 (m, 15H, 3×Ph), 6.98 (s, 1H,CHPh₂), 6.03 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 5.32 (dd, 1H, H-3', J=3and 3.9 Hz), 5.22 and 5.15 (d,d, 1H, 1H, OCH₂ Ph, J=12 Hz), 4.61 (d, 1H,H1', J=1.5 Hz), 4.07 and 3.73 (d,d, 1H, 1H, 8a-CH₂, J=9.3 Hz), 3.85 (m,1H, H-2'), 3.75 (m, 1H, H-5'), 3.27 (dd, 1H, H4', J=3.3 and 9.3 Hz),2.71 (t, 1H, H-1, J=3.9 Hz), 2.32 (d 1H, OH, J=3 Hz), 2.22 (m,1H,CH(CH₃)₂).

INTERMEDIATE 34

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-2-O-(2,2,2-trichloethoxycarbonyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a cold (0° C.) solution of Intermediate 1 (200 mg) and4-dimethylaminopyridine (100 mg) in acetonitrile (10 ml) was addeddropwise a solution of 2,2,2-trichloroethyl chloroformate (50 μl) inacetonitrile (10 ml), and the mixture was stirred at 0° C. until all thestarting material was consumed (tic analysis 2:1 hexane:ethyl acetate).The solvent was removed under vacuum and the residue partitioned betweenethyl acetate (50 ml) and 1N aqueous hydrochloric acid (50 ml). Theorganic layer was washed successively with water and brine, then driedover sodium sulphate, filtered and concentrated. The residue waspurified by flash chromatography on silica gel eluting with hexane:ethylacetate (3:1) to give the title compound (175 mg) as a colourless gumwhich was triturated with hexane to afford a white solid.

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 7.45-7.2 (m, 10H, 2Ph), 6.97 (s, 1H,CHPh₂), 6.04 (dd, 1H, H-2, J=1.2 and 3.6 Hz), 5.03 (dd, 1H, H-2', J=1.5and 4.2 Hz), 4.84, 4.75 (2d, 2H, Cl₃ CCH₂ O, J_(AB) =12 Hz), 4.72 (d,1H, H-1'), 4.2 (m, 1H, H-3'), 4.05, 3.68 (2d, 2H, 8aCH₂, J_(AB) =9 Hz),3.82-3.71 (m, 1H, H-5'), 3.43 (s, 31H, OCH₃), 3.21 (dd, 1H, H4', J=3 and8.4 Hz), 2.70 (m, 1H, H-1), 2.47 (d, 1H, OH3', J=2.1 Hz), 2.28-2.16 (m,1H, CH(CH₃)₂).

INTERMEDIATE 35

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2-O-Benzyloxycarbonyl-3-O-butoxyacetyl-6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 24 (400 mg) in dry dichloromethane (15 ml)at room temperature were added 4-dimethylaminopyridine (67 mg) and asolution of butoxyacetyl chloride (0.1 ml) in dichloromethane (5 ml).After stirring for 1 hour the solvent was removed and the residuepurified by flash column chromatography eluting with hexane:ethylacetate (5:1) to give the title compound (350 mg).

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 7.25-7.43 (m, 15H, 3Ph), 6.96 (s, 1H,CHPh₂), 5.97 (dd, 1H, H-2, J=3.3 and 0.9 Hz), 5.53 (dd, 1H, H-3', J=4.8and 3.3 Hz), 5.18 (AB system, 2H, CH₂ --Ph, J=12 Hz), 4.93 (dd, 1H,H-2', J=1.5 and 4.8 Hz), 4.63 (d, 1H, H-1', J=1.5 Hz), 4.13 (AB system,2H, OCH₂ CO₂, J=14 Hz), 3.66 and 3.99 (2d, 2H, 8aCH₂, J=9 Hz), 3.73 (m,1H, H-5'), 3.53 (m, 2H, CH2O), 3.32 (s, 3H, 4'--OCH₃), 3.24 (dd, 1H,H-4', J=8.4 and 3 Hz).

INTERMEDIATE 36

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2-O-Benzyloxycarbonyl-6-deoxy-4-O-methyl-3-O-octanoyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 24 (600 mg) and 4-dimethylaminopyridine(276 mg) in dry dichloromethane (30 ml) at room temperature was addedoctanoyl chloride (170 μl) and the mixture was stirred for 3 hours. Thesolution was washed with water (2×50 ml), dried over magnesium sulfateand concentrated under reduced pressure. The residue was purified byflash cromatography eluting with hexane:ethyl acetate (4:1) to give thetitle compound (490 mg) as a white foam.

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 7.45-7.25 (m, 15H, 3Ph), 6.97 (s, 1H,CHPh₂), 5.97 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 5.46 (dd, 1H, H-3', J=3and 4.5 Hz), 5.18 (AB system 2H, CH₂ Ph, J=12 Hz), 4.90 (dd, 1H, H-2',J=1.5 and 4.8 Hz), 4.63 (d, 1H, H-1', J=1.8), 3.98 and 3.67 (2d, 2H,8aCH₂, J=8.7 Hz), 3.74 (m, 1H, H-5'), 3.32 (s, 3H, OCH₃), 3.22 (dd, 1H,H4', J=3 and 8.1 Hz), 2.57 (t, 1H, H-1, J=3.9 Hz), 2.37 (dt, 2H, CH₂ CO,J=1.8 and 7.5 Hz), 1.4-1.2 (m, 5CH₂ and 6'CH₃).

INTERMEDIATE 37

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2-O-Benzyloxycarbonyl-6-deoxy-3-O-methoxyacetyl-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 24 (400 mg) in dry dichloromethane (15 ml)at room temperature were added 4-dimethylaminopyridine (70 mg) andmethoxyacetyl chloride (51 μl) in dichloromethane (5 ml). After stirringfor 2 hours the solvent was removed and the residue purified by flashcolumn chromatography eluting with hexane:ethyl acetate (5:1) to givethe title compound (352 mg).

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 7.20-7.42 (m, 15H, 3Ph), 6.96 (s,1Ha, CHPh₂), 5.97 (dd, 1H, H-2, J=3.3 and 0.39 Hz), 5.54 (dd, 1H, H-3',J=4.5 and 3 Hz), 5.18 (AB system, 2H, CH2Ph, J=12 Hz), 4.93 (dd, 1H,H-2', J=4.5 and 1.5 Hz), 4.63 (d, 1H, H-1', J=1.5 Hz), 4.10 (AB system,2H, OCH₂ CO₂, J=12 Hz), 3.66 and 3.98 (2d, 2H, 8aCH₂, J=9 Hz), 3.72 (dq,1H, H-5', J=9 and 6 Hz), 3.34 (s, 3H, 4'OCH₃), 3.45 (s, 3H, CH₃ OCH₂CO₂), 3.24 (dd, 1H, H-4', J=9 and 3 Hz), 2.57 (t, 1H, H-1, J=3.9 Hz).

INTERMEDIATE 38

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-3-O-((E)-2-methyl-2-hexenoyl)-2-O-(2,2,2-trichloroethoxycarbonyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A mixture of trans-2-methyl-2-hexenoic acid (61 mg), triethylamine (167ml), Intermediate 34 (227 mg), 2-chloro-1-methylpyridinium iodide (123mg) and a catalytic amount of 4-dimethylaminopyridine in drydichloromethane (10 ml) was refluxed for 10 minutes. A further quantityof 4-dimethylaminopyridine (88 mg) was added and reflux continued for2.5 hours. The solvent was removed in vacuo and the residue partitionedbetween ethyl acetate (50 ml) and water (50 ml). The organic layer waswashed successively with aqueous hydrochloric acid (1N, 30 ml),saturated aqueous sodium hydrogen carbonate solution (30 ml) and brine(30 ml), then dried (Na₂ SO₄), filtered and concentrated in vacuo. Theresidue was flash chromatographed over silica gel eluting withhexane:ethyl acetate (8:1) and the required fractions combined andevaporated to give the title compound (205 mg) as a white foam.

(¹ H, CDCl₃): 9.72 (s, 1H, CHO), 7.46-7.22 (m, 10H, 2Ph), 6.98 (s, 1H,CHPh₂), 6.82 (m, 1H, RO₂ C(CH₃)=CH--R), 6.03 (m, 1H, H-2), 5.54 (m, 1H,H-3'), 5.0 (m, 1H, H-2'), 4.84, 4.76 (2d, 2H, Cl₃ CCH₂, J_(AB) =12 Hz),4.67 (d, 1H, H-1', J=1.5 Hz), 4.05, 3.71 (2d, 2H, 8aCH₂, J_(AB) =9 Hz),3.8 (m, 1H, H-5'), 3.37 (s, 3H, --OCH₃), 3.32 (m, 1H, H4'), 2.68 (bt,1H, H-1, J=3.6 Hz), 2.3-2.12 (m, 3H, CHMe₂ and RO₂ CC(CH₃)=CH--CH₂ --R).

INTERMEDIATE 39

[1R-(1α, 3aβ, 4β, 4aβ7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-3-O-((E)-2-methyl-2-hexenoyl)-2-O-(2,2,2-trichloroethoxycarbonyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A solution of Intermediate 38 (194 mg) in a (20:1) trifluoroaceticacid:water mixture (5 ml) at 0° C. was stirred for 45 minutes. Thesolvent was removed in vacuo and the residue co-evaporated with toluene(2×10 ml) to a yellow oil. This was purified by preparative tic (Merck5717) eluting with dichloromethane:methanol (20:1) and washing theproduct off the silica gel with ethyl acetate:methanol (6:1). Removal ofsolvent gave the title compound (138 mg) as a white foam.

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 6.81 (m, 1H, RO₂ CC(CH₃)=CH--R), 6.05(dd, 1H, H-2, J=1.5 and 3.6 Hz), 5.55 (dd, 1H, H-3', J=3 and 4.5 Hz),5.02 (dd, 1H, H-2', J=1.5 and 4.5 Hz), 4.81 (m, 2H, Cl₃ CCH₂), 4.70 (d,1H, H-1', J=1.5 Hz), 4.17, 3.57 (2d, 2H, 8aCH₂, J_(AB) =9 Hz), 3.8 (m,1H, H-5'), 3.36 (s, 3H, --OCH₃), 3.31 (m, 1H, H-4'), 2.59 (m, 1H, H-1),2.32 (m, 1H, CHMe₂), 2.17 (m, 2H, RO₂ C--C(CH₃)=CH--CH₂ --R).

INTERMEDIATE 40

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2-O-Benzyloxycarbonyl-3-O-(4-chlorobutyryl)-6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 24 (500 mg) and 4-dimethylaminopyridine(457 mg) in dry dichloromethane (25 ml) at room temperature was added4chlorobutyryl chloride (141 μl) and the mixture was stirred for 24hours. The solution was washed with water (2×50 ml), dried overmagnesium sulfate, and concentrated under reduced pressure. The residuewas purified by flash cromatography eluting with hexane:ethyl acetate(3:2) to give the title compound (360 mg) as a white foam.

δ (¹ H, CDCl₃): 9.7 (s, 1H, CHO), 7.45-7.24 (m, 15H, 3Ph), 6.97 (s,1H,CHPh₂), 5.97 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 5.46 (dd, 1H, H-3', J=3and 4.5 Hz), 5.18 (AB system, 2H, CH₂ Ph, J=12 Hz), 4.91 (dd, 1H, H-2',J=1.5 and 4.5 Hz), 4.64 (d,1 H, H-1', J=1.8 Hz), 3.99 and 3.67 (2d, 2H,8aCH₂, J=8.7 Hz), 3.75 (dd, 1H, H-5', J=6.3 and 8.4 Hz), 3.60 (t, 2H,CH₂ CO, J=6.6 Hz), 3.26 (s, 3H, OCH,₃), 3.22 (dd, 1H, H-4', J=3.0 and8.4 Hz), 2.58 (m, 3H, 1 H, H-1 and 2H, CH₂ Cl).

INTERMEDIATE 41

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aβ, 8aβ)]8a-[(2-O-Benzyloxycarbonyl-6-deoxy-4-O-methyl-3-O-(2-methylpropanoyl)-.beta.-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of isobutyryl chloride (0.054 ml) in dichloromethane (5 ml)was added to a mixture of Intermediate 24 (0.415 g) and4-dimethylaminopyridine (0.064 g) in dichloromethane (10 ml). Thereaction mixture was stirred for 20 hours. Water (15 ml) was added intothe mixture and the organic layer was separated. The solvent was removedin vacuo and the residue was chromatographed on silica gel flash columneluting with hexane:ethyl acetate (3:1) to give the title compound(0.276 g).

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 7.30 (m, 15H, 3×Ph), 6.97 (s, 1H, Ph₂CH), 5.97 (dd, 1H, H-2, J=1.2 and 3.6 Hz), 5.40 (dd, 1H, H-3', J=3.0 and4.5 Hz), 5.18 (AB system, 2H, PhCH₂ OCO), 4.90 (dd, 1H, H-2', J=1.5 and4.8 Hz), 4.62 (d, 1H, H-1, J=1.8 Hz), 3.98 (d, 1H, 8aCH₂, J_(AB) =9.0Hz), 3.75 (m, 1H, H-5'), 3.67 (d, 1H, 8aCH₂, J_(AB) =9.0 Hz), 3.32 (s,3H, OMe), 3.22 (dd, 1H, H-4', J=3.0 and 8.1 Hz), 2.62 (m, 2H, H-1 and(CH₃)₂ CHCO₂).

INTERMEDIATE 42

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8β)]8a-[(2-O-Benzyloxycarbonyl-6-deoxy-4-O-methyl-3-O-propionyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

Propionic acid (54 μl), 2-chloro-1-methylpyridinium iodide (184 mg),triethylamine (250 μl), Intermediate 24 (285 mg),4-dimethylaminopyridine (132 mg) and dry dichloromethane (20 ml) weremixed and stirred at room temperature for 18 hours. The solvent wasremoved in vacuo and the residue chromatographed on silica gel elutingwith hexane:ethyl acetate (4:1). The appropriate fractions were combinedand the solvent was evaporated to give the title compound (285 mg) as awhite foam.

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 7.44-7.22 (m, 15H, 3Ph), 6.96 (s, 1H,CHPh₂), 5.97 (dd, 1H, H-2, J=1.5 and 3.3 Hz), 5.46 (dd, 1H, H-3', J=3and 4.8 Hz), 5.22, 5.14 (2d, 2H, PhCH2O, J_(AB) =12 Hz), 4.91 (dd, 1H,H-2', J=1.5 and 4.8 Hz), 4.63 (d, 1H, H-1', J=1.5 Hz), 3.99, 3.67 (2d,2H, 8aCH₂, J_(AB) =8.7 Hz), 3.75 (m, 1H, H-5'), 3.33 (s, 3H, --OCH₃),3.23 (dd, 1H, H-4', J=3 and 8.4 Hz), 2.57 (m, 1H, H-1), 2.4 (dq, 2H, RO₂CCH₂ --Me, J=7.5 Hz (q) and 1.2 Hz (d)), 2.22 (m, 1H, CHMe₂),1.16 (t,3H, RO₂ CCH₂ CH₃, J=7.5 Hz).

INTERMEDIATE 43

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2-O-Benzyloxycarbonyl-6-deoxy-4-O-methyl-3-O-(trans-4-methyl-1-cyclohexanecarbonyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A mixture of trans-4-methyl-1-cyclohexanecarboxylic acid (102 mg),2-chloro-1-methylpyridinium iodide (184 mg), triethylamine (250 μl),Intermediate 24 (285 mg) and 4-dimethylaminopyridine (132 mg) in drydichloromethane (20 ml) was stirred at room temperature for 15 hours.The solvent was removed in vacuo and the residue purified by columnchromatography on silica gel eluting with hexane:ethyl acetate (4:1).The required fractions were combined and evaporated to give the titlecompound (310 mg) as a white foam.

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 7.44-7.22 (m, 15H, 3Ph), 6.97 (s, 1H,CHPh₂), 5.97 (dd, 1H, H-2, J=1.2 and 3.3 Hz), 5.43 (dd, 1H, H-3', J=3.3and 4.8 Hz), 5.21, 5.14 (2d, 2H, Ph-CH₂ --O, J_(AB) =12 Hz), 4.89 (dd,1H, H-2', J=1.8 and 4.8 Hz), 4.62 (d, 1H, H-1', J=1.8 Hz), 3.99, 3.68(2d, 2H, 8aCH₂, J_(AB) =9 Hz), 3.75 (m, 1H, H-5'), 3.31 (s, 3H, OCH₃),3.21 (dd, 1H, H4', J=3.3 and 8.4 Hz), 2.57 (m, 1H, H-1), 2.34-2.16 (m,2H, CHMe₂ +RO₂ C--CH--(cyclohexane)), 0.89 (d, 3Haprox, H₃C-cyclohexane-CO₂ R).

INTERMEDIATE 44

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-trans-Cinnamoyl-6-deoxy-4-O-methyl-2-O-(2,2,2-trichloroethoxycarbonyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A cold (0° C.) solution of Intermediate 34 (75 mg) in drydichloromethane (4 ml) under nitrogen was treated with4-dimethylaminopyridine (50 mg) and trans-cinnamoyl chloride (30 mg).The mixture was stirred at room temperature until all the startingmaterial was consumed (5 hours). The reaction was quenched with waterand the mixture stirred for 15 minutes and then partitioned betweenethyl acetate (50 ml) and 1N aqueous hydrochloric acid (50 ml). Theorganic layer was washed successively with water and brine, then dried(Na₂ SO₄), filtered and concentrated. The residue material was flashchromatographed using hexane to 3:1 hexane:ethyl acetate as eluent togive the title compound (90 mg).

δ (¹ H, CDCl₃): 9.72 (s, 1H, CHO), 7.74 (d, 1H, PhCH=CH--CO₂ R, J=15.9Hz), 7.6-7.2 (m, 15H, 3Ph), 6.98 (s, 1H, CHPh₂), 6.49 (d, 1H,PhCH=CH--CO₂ R), 6.04 (dd, 1H, H-2, J=1.2 and 3.6 Hz), 5.60 (dd, 1H,H-3', J=3.3 and 4.5 Hz), 5.06 (dd, 1H, H-2', J=1.5 and 4.5 Hz), 4.9-4.7(m, 3H, Cl₃ CCH₂ O, H-1'), 4.07, 3.72 (2d, 2H, 8aCH₂, J_(AB) =9 Hz),3.94-3.82 (m, 1H, H-5'), 3.44-3.32 (m, 4H, --OCH₃, H4'), 2.7 (t, 1H,H-1, J=3.6 Hz), 2.3-2.18 (m, 1H, --CHMe₂).

INTERMEDIATE 45

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-trans-Cinnamoyl-6-deoxy-4-O-methyl-2-O-(2,2,2-trichlorethoxycarbonyl)-62-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A suspension of Intermediate 44 (295 mg) in trifluoroacetic acid:water(4:1;10 ml) at 0° C. was stirred for 1.5 hours. The solvent was removedin vacuo and the crude co-evaporated with toluene (3×5 ml). The residuewas flash chromatographed on silica gel eluting withdichloromethane:methanol (99:1 to 95:5) and appropriate fractions werecombined and concentrated to give the title compound (200 mg).

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 7.74 (d, 1H, Ph--CH=CH--CO₂ R, J=15.9Hz), 7.6-7.36 (m, 5H, Ph), 6.48 (d, 1H, Ph--CH=CH--CO₂ R, J=15.9 Hz),6.06 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 5.61 (dd, 1H, H-3', J=3.3 and 4.5Hz), 5.07 (dd, 1H, H-2', J=1.5 and 4.5 Hz), 4.82 (s, 2H, Cl₃ CCH₂ O),4.77 (d, 1H, H-1', J=1.5 Hz), 4.15, 3.61 (2d, 2H, 8aCH₂, J_(AB) =9 Hz),3.95-3.80 (m, 1H, H-5'), 3.40 (s, 3H, --OCH₃), 3.35 (dd, 1H, H-4', J=3.3and 8.4 Hz), 2.62 (m, 1H, H-1), 2.32 (m, 1H, --CHMe₂).

INTERMEDIATE 46

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-3-Omethacryloyl-4-O-methyl-2-O-(2,2,2-trichloroethoxycarbonyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A mixture of Intermediate 34 (100 mg), triethylamine (80 μl),methacrylic acid (21 μl), 2-chloro-1-methylpyridinium iodide (68 mg) anda catalytic amount of 4-dimethylaminopyridine in dry dichloromethane (10ml) was refluxed under nitrogen for 5 minutes. A further quantity of4-dimethylaminopyridine (45 mg) was added and reflux was continued untilall the starting material was consumed (tic control 4:1 hexane:ethylacetate). The solvent was removed in vacuo and the residue partitionedbetween ethyl acetate (50 ml) and 1N aqueous hydrochloric acid (50 ml).The organic layer was washed successively with saturated aqueous sodiumhydrogen carbonate solution, water and brine, then dried (Na₂ SO₄),filtered and concentrated in vacuo. The residue was purified by flashchromatography eluting with hexane:ethyl acetate (4:1) to give the titlecompound (60 mg).

δ (¹ H, CDCl₃): 9.72 (s, 1H, CHO), 7.46-7.22 (m, 10H, 2Ph), 6.98 (s, 1H,Ph₂ CH--), 6.16 (bs, 1H, Ha--C=C), 6.03 (m, 1H, H-2), 5.65 (m, 1H,Hb--C=), 5.53 (dd, 1H, H-3', J=3 and 4.8 Hz), 5.01 (dd, 1H, H-2', J=1.5and 4.8 Hz), 4.84, 4.76 (2d, 2H, Cl₃ CCH₂, J_(AB) =11.7 Hz), 4.67 (d,1H, H-1', J=1.5 Hz), 4.04, 3.7 (2d, 2H, 8aCH₂, J_(AB) =9 Hz), 3.8 (m,1H, H-5'), 3.36 (s, 3H, --OCH₃), 3.31 (dd, 1H, H4', J=3.3 and 8.4 Hz),2.68 (m, 1H, H-1), 2.23 (m, 1H, CHMe₂), 1.97 (s, 3H, H₃ C--C=).

INTERMEDIATE 47

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-3-O-methacryloyl-4-O-methyl-2-O-(2,2,2-trichloroethoxycarbonyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A suspension of Intermediate 46 (150 mg) in trifluoroacetic acid:water(4:1;7 ml) at 0° C. was stirred for 1.5 hours. The solvent was removedin vacuo and the residue co-evaporated with toluene (3×3 ml). Theresidue was flash chromatographed on silica gel eluting withdichloromethane:methanol (25:1) and appropriate fractions were combinedand evaporated to give the title compound (115 mg) as a white foam.

δ (¹ H, CDCl₃): 9.72 (s, 1H, CHO), 6.15 (m, 1H, Ha--C=C), 6.05 (dd, 1H,H-2, J=1.5 and 3.6 Hz), 5.65 (m, 1H, Hb--C=C), 5.54 (dd, 1H, H-3', J=3and 4.5 Hz), 5.02 (dd, 1H, H-2', J=1.8 and 4.5 Hz), 4.81 (s, 2H, Cl₃CCH₂), 4.70 (d, 1H, H-1', J=1.8 Hz), 4.1, 3.59 (2d, 2H, 8aCH₂, J_(AB) =9Hz), 3.81 (m, 1H, H-5'), 3.36 (s, 3H, --OCH₃), 3.31 (dd, 1H, H4', J=3and 8.4 Hz), 2.62 (m, 1H, H-1), 2.31 (m, 1H, CHMe₂), 1.97 (s, 3H, H₃C--C=C).

INTERMEDIATE 48

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2-O-Benzyloxycarbonyl-6-deoxy-4-O-methyl-3-O-octyloxycarbonyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 24 (400 mg) and 4-dimethylaminopyridine(238 mg) in dry dichloromethane (50 ml) at room temperature was addedoctyl chloroformate (127 μl) and the mixture was stirred for 3 hours.The solution was washed with water (2×50 ml), dried over magnesiumsulfate, and concentrated under reduced pressure. The residue waspurified by flash chromatography eluting with hexane:ethyl acetate (4:1)to give the title compound (320 mg) as a white foam.

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 7.45-7.20 (m, 15H, 3Ph), 6.96 (s, 1H,CHPh₂), 5.97 (dd, 1H, H-2, J=1.2 and 3.3 Hz), 5.26 (dd, 1H, H-3', J=3and 4.2 Hz), 5.18 (AB system, 2H, CH₂ --Ph, J=12 Hz), 5.00 (dd, 1H,H-2', J=1.5 and J=4.2 Hz), 4.69 (d, 1H, H-1', J=1.5 Hz), 4.15 (t, 2H,CH₂ OCO, J=6.6 Hz), 4.00 and 3.66 (2d, 2H, 8aCH₂, J=8.7 Hz), 3.78 (dd,1H, H-5', J=6 and 9 Hz), 3.37 (s, 3H, OCH₃), 3.30 (dd, 1H, H4', J=3 and8.4 Hz).

INTERMEDIATE 49

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-2-O-benzyloxycarbonyl-4-O-methyl-3-O-octylaminocarbonyl-.beta.-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 24 (250 mg) and octyl isocyanate (62 mg)in dry toluene (10 ml) was added a catalytic amount (5 drops) ofdibutyltin diacetate, and the mixture was refluxed under nitrogen for 1hour. The solvent was evaporated to dryness and the residuechromatographed on a silica gel flash column using hexane:ethyl acetate(7:3) as eluent to give the title compound (280 mg) as a white foam.

δ (¹ H, CDCl₃): 9.70 (s, 1H, CHO), 6.96 (s. 1H, CHPh₂), 5.96 (dd, 1H,H-2, J=1.5 and 3.6 Hz), 5.32 (m, 1H, H-3'), 5.17 (AB system, 2H, --CH₂OPh, J=12.0 Hz), 4.98 (dd, 1H, H-2', J=1.8 and 5.1 Hz), 4.75 (t, 1H,--NH--C=O--), 4.62 (d, 1H, HI-1', J=1.8 Hz), 3.98 and 3.65 (2d, 2H,8a-CH2, J=9 Hz), 3.74 (m, 1H, H-5'), 3.37 (s, 3H, 4'--OCH3), 3.25 (dd,1H, H-4', J=3 and 8.1 Hz), 3.18 (m, 2H, --CH₂ --NH--CO₂), 2.58 (t, 1H,H-1').

INTERMEDIATE 50

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-3-O-benzylaminocarbonyl-2-O-benzyloxycarbonyl-4-O-methyl-.beta.-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a solution of Intermediate 24 (240 mg) and benzyl isocyanate (54 mg)in dry toluene (10 ml) was added a catalytic amount of dibutyltindiacetate, and the stirred mixture was refluxed under nitrogen for 6hours. The solvent was evaporated to dryness and the residuechromatographed on a silica gel flash column eluting with hexane:ethylacetate (5:1) to give the title compound (140 mg) as a white foam.

δ (¹ H, CDCl₃): 9.70 (s, 1H, CHO), 6.95 (s, 1H, CHPh₂), 5.96 (dd, 1H,H-2, J=0.6 and 2.7 Hz), 5.37 (dd, 1H, H-3', J=3 and 4.8 Hz), 5.18 (ABsystem, 2H, O--CHPh₂, J=12 Hz), 5.12 (t, 1H, CH₂ --NH--CO₂), 4.98 (dd,1H, H-2', J=0.3 and 1.2 Hz), 4.61 (d, 1H, H-1', J=1.2 Hz), 4.38 (m, 2H,NH--CH₂ --Ph), 3.98 and 3.64 (2d, 2H, 8a-CH₂, J=9 Hz), 3.74 (m, 1H,H-5'), 3.38 (s, 3H, 4'--OCH), 3.26 (dd, 1H, H-4', J=3 and 8.1 Hz), 2.57(t, 1H, H-1).

INTERMEDIATE 51

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2-O-Benzyloxycarbonyl-6-deoxy-3-O-dimethylaminocarbonyl-4-O-methyl-.beta.-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

A solution of Intermediate 24 (0.500 g) in tetrahydrofuran (10 ml) wasadded via cannula to a solution of lithium diisopropylamide solution intetrahydrofuran (5 ml) [prepared from duisopropylamine (0.115 ml) and2.5M n-butyllithium in hexane (0.33 ml) at 0° C.]. Dimethylaminocarbonylchloride (0.06 ml) was then added and the reaction mixture was stirredat 0° C. for 1 hour and then maintained at room temperature for 18hours. The mixture was diluted with ethyl acetate (20 ml) and water (10ml) and the organic layer was separated and dried (sodium sulfate). Thesolvent was evaporated in vacuo and the residue was chromatographed on asilica gel flash column eluting with hexane:ethyl acetate (2:1) to givethe title compound (0.171 g).

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 7.43 to 7.26 (m,15H, 3×Ph), 6.96 (s,1H, Ph₂ CH), 5.97 (dd, 1H, H-2, J=1.5 and 3.6 Hz), 5.35 (dd, 1H, H-2',J=3.0 and 4.8 Hz), 5.17 (m, 2H, PhCH₂ OCO), 4.98 (dd, 1H, H-3', J=1.5and 4.8 Hz), 4.61 (d, 1H, H-1', J=1.8 Hz), 3.97 (d, 1H, 8aCH₂, J_(AB)=9.0 Hz), 3.75 (m, 1H, H-5'), 3.66 (d, 1H, 8aCH₂, J_(AB) =9.0 Hz), 3.36(s, 3H, OMe), 3.25 (dd, 1H, H4', J=5.1 and 3.0 Hz), 2.93 (s, 3H, CH₃ N),2.91 (s, 3H, CH₃ N), 2.58 (m, 1 H, H-1).

INTERMEDIATE 52

(a) [1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,3,6,-Trideoxy-2,3-didehydro-4-O-methyl-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester and

(b) [1R-(1α, 3aβ, 4β, 4aβ, 7β7aα, 8aβ)]8a-[(3,6-Dideoxy-3-iodo-4-O-methyl-β-altro andmannopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl esters

Intermediate 1 (1.5 g), triphenylphosphine (2.4 g) and imidazole (640mg) were refluxed in toluene (50 ml) with stirring and zinc (40 mg) wasadded. Then, iodine (1.75 g) was added in small portions. The reactionmixture was refluxed for 4 hours, cooled, decanted into 10% aqueoussodium hydrogen carbonate (50 ml) and 5% aqueous sodium thiosulfate (20ml) and then shaken until the iodine was consumed. The toluene phase wasextracted with water, dried over sodium sulfate and concentrated. Theresidue was purified by flash column chromatography eluting withhexane:ethyl acetate (10:1 to 6:1) to afford title compound (a) with thehigher Rf as a colourless syrup (185 mg) and title compound (b) (as amixture of 3'-iodo isomers) with the lower Rf as a pale yellow foam (375mg).

(a) δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 7.24-7.45 (m, 10H, 2Ph), 6.99 (s,1H, CHPh₂), 6.01-6.09 (m, 2H, H-2 and H-3'), 4.97 (dd, 1H, H-1', J=1.8and 3.6 Hz), 3.74 and 4.00 (2d, 2H, 8aCH₂, J=9.3 Hz), 3.64 (m, 1H,H-5'), 3.49 (m, 1H, H4'), 3.40 (s, 3H, 4'--OCH₃).

(b) δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 7.26-7.45 (m, 10H, 2Ph), 6.98 and6.99 (2s, 1H, CHPh₂ of both isomers), 6.03 and 6.08 (2dd, 1H, H-2 ofboth isomers), 4.35 and 4.99 (2s, 1H, H-1' of each isomer), 4.75 (t, 1H,H-3' of isomer B), 4.14 (m, 1H, H-2' of isomer B), 4.06 (m, 1H, Ha-8aCH₂of both isomers and 1H, H3' of isomer A), 3.75 (m, 1H, Hb-8aCH₂ of bothisomers), 3.72 (m, 1H, H-5' of isomer B), 3.34 and 3.56 (2s, 3H,4'--OCH₃), 3.30 (m, 2H, H-4' and H-5' of isomer A), 2.57 (m, 1H, H4' ofisomer B).

INTERMEDIATE 53

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3,6-Dideoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

To a degassed solution of Intermediate 52(b) (115 mg) in toluene (15 ml)under nitrogen, tributyltin hydride (0.08 ml) andazobis(isobutyronitrile) (15 mg) were added. After 1 hour under reflux,the solvent was removed in vacuo to give a syrup, which was purified byflash chromatography eluting with hexane:ethyl acetate (6:1) to yieldthe title compound (62 mg).

δ (¹ H, CDCl₃): 9.74 (s, 1H, CHO), 7.26-7.44 (m, 10H, 2Ph), 6.99 (s, 1H,CHPh₂), 6.04 (dd, 1 H, H-2, J=1.2 and 3.3 Hz), 4.32 (d, 1H, H-1', J=1.2Hz), 3.76 and 4.08 (2d, 2H, 8aCH₂, J=9 Hz), 3.87 (m, 1H, H-2'), 3.36 (s,3H, 4'--O--CH₃), 3.34 (m, 1H, H-5'), 3.21 (dq, 1H, H4', J=13.2 and 4.5Hz), 2.74 (t, 1H, H-1, J=3.6 Hz), 2.38-2.45 (m, 1H, H-3'a), 2.23 (m, 2H,CHMe₂ and 2'--OH).

INTERMEDIATE 54

[1R-(1α, 3aβ, 4b, 4aβ, 7β, 7aα, 8aβ)]8a-(((2,3,6-Trideoxy-4-O-acetyl-3-acetylthio-β-D-allopyranosyl)oxy)methyl)-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

Cesium thioacetate (1.4 mmol) was prepared in situ, by addition ofcesium carbonate (494 mg) to a solution of thioacetic acid (0.1 ml) indry methanol (5 ml). After 30 minutes, the solvent was stripped off andthe crude product was dissolved in dry dimethylformamide (5 ml). Theintermediate 21 (500 mg) was added to this solution, dissolved in 3 mlof dimethylformamide. The reaction mixture was stirred at roomtemperature for 2 days, and after this time, the solvent was evaporatedin vacuo, and the crude product purified by flash column chromatographyon silica gel, eluting with hexane:ethyl acetate 3:1 to give pure titlecompound (100 mg).

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 7.33 (m, 10H, 2Ph), 6.98 (s, 1H,CH--Ph₂), 6.05 (d, 1 H, H-2, J=2.4 Hz), 4.75 (dd, 1H, H-1', J=8.1 and3.9 Hz), 4.43 (dd, 1H, H-3', J=5.1 and 3.9 Hz), 4.31 (dd, 1H, H4', J=8.7and 4.5 Hz), 3.99 (d, 1H, H-8a, J=9.3 Hz), 3.72 (dq, 1H, H-5', J=8.1 and6.0 Hz), 3.67 (d, 1H, H-8a, J=9.3 Hz), 2.72 (t, 1H, H-1, J=3.9 Hz), 2.35(s, 3H₃), 2.24 (m, 1H, --CH(CH₃)₂), 2.00 (s, 3H, CH₃).

INTERMEDIATE 55

[1R-(1α, 3aβ, 4b, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-)-benzoyl-2,6-dideoxy-4-O-methyl-β-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

Intermediate 10 (200 mg) and dimethylaminopyridine (126 mg) were solvedin 20 ml of dry dichloromethane. Benzoyl chloride (146 mg) in 5 ml ofdry dichloromethane was added dropwise and the reaction mixture wasrefluxed for 6 hours. The mixture of reaction was washed with asaturated solution of sodium bicarbonate (50 ml), water (50 ml) andbrine (50 ml), dried over magnesium sulphate and evaporated to dryness.The residue was chromatographied with mixtues of hexane:ethyl acetate togive the title compound (180 mg, 78% yield) as a white foam.

δ (1H, CDCl₃): 9.72 (s, 1H, CHO), 8.07, 7.6-7.2 (dd, m, 2H, 13H,Ph--CO+, PL2--CH), 6.99 (s, 1H, Ph₂ CH), 6.02 (dd, 1H, H-2, J-3.6 and1.9 Hz), 5.7 (c, 1H, H-3'), 4.7 (dd, 1H, H-1'), 4.04, 3.71 (dd, 1H, 1H,8aCH₂, 8bCH₂, J=10 Hz), 3.9-3.8 (m, 1H, H-5'), 3.36 (s, 3H, OCH₃), 3.04(dd, 1H, H4', J=3.6 and 7.5 Hz), 2.7 (t, 1H, H-1).

INTERMEDIATE 56

[1R-(1α, 3aβ, 4b, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-acetyl-2,6-dideoxy-4-O-methyl-β-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid, diphenylmethyl ester

Intermediate 10 (150 mg), acetic anhydre (0.12 ml, 120 mg) anddimethylaminopyridine (141 mg) were solved in dry dichloromethane (25ml) and stirred overnight at room temperature under nitrogen atmosphere.The solvent was eliminated and the residue purified by flashchromatography with hexane:ethyl acetate 9:1, to give the title compound(140 mg, 85% yield) as a white foam.

δ (¹ H, CDCl₃): 9.74 (s, 1H, CHO), 7.45, 7.24 (m, 10H, (Ph)₂ CH)), 6.98(s, 1H, (Ph)₂ CH)), 6.05 (dd, 1H, H-2, J=1.8 and 2.16 Hz), 5.51 (c, 1H,H-3'), 4.55 (dd, 1H, H-1', J=0.6 and 7.2 Hz), 4.02, 3.6 (d, d, 1H, 1H,8aCH₂, J=10.8 Hz), 3.72 (m, 1H, H-5'), 3.34 (s, 3H, OCH₃), 2.90 (dd, 1H,H4'), 2.73 (t, 1H, H-1), 2.11 (s, 3H, CH₃ CO).

EXAMPLE 1

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-3-O-(3-phenylprop-2(E)-enyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A solution of sordarin (1 mmol) in dry tetrahydrofuran (20 ml) undernitrogen was treated with sodium hydride (3.8 mmol). After 30 minutes, asolution of cinnamyl bromide (1.1 mmol) in dry tetrahydrofuran (5 ml)was added. The mixture was stirred for 3 days, and then diluted withethyl acetate and washed with water. The organic phase was evaporatedand the residue purified by flash chromatography eluting withdichloromethane:methanol (30:1) to give the title compound (73 mg).

δ (¹ H, CDCl₃): 9.80 (s, 1H, CHO), 7.33 (m, 5H, Ph), 6.58 (d, 1H, H-3",J=15.9 Hz), 6.31 (m, 1H, H-2"), 6.00 (dd, 1H, H-2, J=1.2 and 3.3 Hz),4.66 (d, 1H, H-1', J=1.2 Hz), 4.36 (m, 2H, CH₂ --1"), 4.24 and 3.47 (d,d1H, 1H, 8a-CH₂, J=9.3 Hz), 4.17 (t, 1H, H-3', J=3.9 Hz), 3.68 (m, 1H,H-2'), 3.65 (m, 1H, H-5'), 3.19 (dd, 1H, H4', J=3.3 and 9.3 Hz); δ (¹³C, CDCl₃): 204.7 (CHO), 175.1 (CO₂ H), 148.3 (C-3), 136.6, 133.0, 128.6,128.5, 127.7, 126.5 and 126.2 (Ph, C-2" and C-3"), 130.4 (C-2), 98.8(C-1'), 80.0 (C-4'), 75.8 (C-5'), 74.3 (8a-CH₂), 73.3 (C-3a), 73.0(C-2"), 68.5 (C-3'), 66.5 (C-2').

EXAMPLE 2

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-Benzyl-6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A stirred solution of Intermediate 2 (0.3mmol) in water (3 ml) at 0° C.was treated with trifluoroacetic acid (7 ml). The mixture was stirredfor 30 minutes and then poured into water:ether (1:1; 50 ml). Theorganic phase was evaporated and the residue purified by flashchromatography using dichloromethane:methanol (20:1) as eluent to givethe title compound (115 mg).

δ (¹ H, CDCl₃): 9.81 (s, 1H, CHO), 7.36 (m, 5H, Ph), 6.03 (dd, 1H, H-2,J=1.2 and 3.6 Hz), 4.86 and 4.59 (AB system, 1H, 1H, CH₂ Ph, J=12.6 Hz),4.66 (d, 1H, H-1', J=0.9 Hz), 4.21 and 3.50 (d,d, 1H, 1H, 8a-CH₂, J=9Hz), 4.05 (t, 1H, H-3', J=3.6 Hz), 3.65 (m, 2H, H-2' and H-5'), 3.19(dd, 1H, H-4', J=3.3 and 9.3 Hz), 2.57 (t, 1H, H-1, J=3.6 Hz), 2.32 (m,1H, CH(CH₃)₂); δ (¹³ C, CDCl₃): 204.7 (CHO), 175.1 (CO₂ H), 148.4 (C-3),138.2 (Cipso), 130.3 (C-2), 128.2, 127.9, 127.6 (Ph), 98.7 (C-1'), 79.8(C-4'), 75.8 (C-5'), 74.3 and 74.1 (8a-CH₂ and CH₂ Ph), 65.6 (C-8a),66.3 (C-3'), 65.3 (C-2'), 57.3 (OCH₃).

EXAMPLE 3

[1R-(1α, 3aβ, 4β4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-3-O-hexyl-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 3 (0.14 mmol) in methanol (20 ml) wasadded 10% palladium on charcoal (20 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 6 psi of hydrogen for 30 minutes atroom temperature. The catalyst was filtered off and the solventevaporated to dryness. The residue was purified by flash chromatographyusing dichloromethane:methanol (25:1) as eluent to give the titlecompound (64 mg).

δ (¹ H, CDCl₃): 9.83 (s, 1H, CHO), 6.03 (d, 1H, H-2, J=3 Hz), 4.64 (d,1H, H-1', J=1.2 H), 4.28 and 3.41 (d,d, 1H, 1H, 8a-CH₂, J=9.3 Hz), 4.14(t, 1H, H-3', J=3.6 Hz), 3.64 (m, 2H, H-2' and H-5'), 3.52 (m, 2H, OCH₂CH₂), 3.15 (dd, 1H, H4', J=3 and 9.3 Hz), 2.52 (m, 1H, H-1), 2.31 (m,11H, CH(CH₃)₂); δ (¹³ C, CDCl₃): 204.7 (CHO), 174.3 (CO₂ H), 148.7(C-3), 130.2 (C-2), 98.4 (C-1'), 80.0 (C-4'), 76.9 (C-5'), 73.9(8a-CH₂), 72.9 (OCH₂ CH₂), 68.5 (C-3'), 66.3 (C-2'), 65.0 (C-8a), 59.0(OCH₃).

EXAMPLE 4

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-2,3,4-tri-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 4 (0.26 mmol) in methanol (20ml) was added10% palladium on charcoal (20 mg) under nitrogen. The mixture was shakenin a Parr apparatus under 8 psi of hydrogen for 30 minutes at roomtemperature. The catalyst was filtered off and the solvent evaporated todryness. The residue was purified by flash chromatography usingdichloromethane:methanol (20:1) as eluent to give the title compound (98mg).

δ (¹ H, CDCl₃): 9.85 (s, 1H, CHO), 6.04 (dd, 1H, H-2, J=1.2 and 3.3 Hz),4.60 (d, 1H, H-1', J=1.5 Hz), 4.33 (d, 1H, 8a-CHa, J=9.3 Hz), 3.71 (m,2H, H-2', and H-3'), 3.52 (s, 3H, 2'--OCH₃), 3.50 (s, 3H, 3'--OCH₃),3.41 (m, 5H, 4'--OCH₃, H-5' and 8a-CHb), 3.16 (dd, 1H, H-4', J=3 and 9.3Hz), 2.50 (t, 1H, H-1, J=3.9 Hz), 2.32 (m, 1H, CH(CH₃)₂).

EXAMPLE 5

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,6-Dideoxy-3,4-O-dimethyl-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

Sodium hydride (24 mg) was added portionwise to a solution ofIntermediate 10 (150 mg) in anhydrous tetrahydrofuran (3 ml) undernitrogen atmosphere at 0° C. The resulting suspension was stirred for 15minutes and methyl iodide (125 μl) was added. After 2 hours of stirring1N ammonium chloride (10 ml) and ethyl acetate (20 ml) were added. Theaqueous phase was extracted with ethyl acetate (2×20 ml) and the organicphase was washed with brine, dried (magnesium sulphate) and evaporatedto dryness. The residue was dissolved in ethyl acetate (10 ml) and 10%palladium on charcoal (100 mg) was added. The mixture was stirred under30 psi of hydrogen for 45 minutes. The catalyst was filtered off and theresidue flash chromatographed eluting with dichloromethane:methanol(30:1) to obtain the title compound (78 mg).

δ (¹ H, CDCl₃): 9.86 (s, 1H, CHO), 6.04 (dd, 1H, H-2, J=1.5 and 3.6 Hz),4.65 (dd, 1 H, H-1', J=1.8 and 9.6 Hz), 4.41 (d, 1H, 8a-CH₂, J=9.3 Hz),3.83-3.70 (m, 2H, H-3' and H-4'), 3.44 (s, 3H, CH₃ O), 3.39 (s, 3H, CH₃O), 3.30 (d, 1H, 8aCH₂, J=9.3 Hz), 2.85 (dd, 1H, H-4', J=3.9 and 9.3Hz), 2.45 (t, 1H, H-1, J=3.6 Hz); δ (¹ H, CDCl₃): 204.5 (CHO), 173.8(CO₂ H), 184.4 (C-3), 130.5 (C-2), 97.5 (C-1'), 82.4 (C-4'), 73.3(8aCH₂), 73.0 (C-3'), 69.5 (C-5'), 65.0 (C8a), 59.0 (C-4), 57.8 (CH₃ O),57.1 (CH₃ O).

EXAMPLE 6

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,6-Dideoxy-3-O-ethyl-4-O-methyl-62-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

Sodium hydride (24 mg) was added portionwise to a solution ofIntermediate 10 in anhydrous tetrahydrofuran under nitrogen atmosphereat 0° C. The resulting suspension was stirred for 15 minutes and ethyliodide (160 μl) was added. After 2 hours of stirring 1N ammoniumchloride (10 ml) and ethyl acetate (20 ml) were added. The aqueous phasewas extracted with ethyl acetate (2×20 ml) and the organic phase waswashed with brine, dried (magnesium sulphate) and evaporated to dryness.The residue was dissolved in ethyl acetate (10 ml) and 10% palladium oncharcoal (100 mg) was added. The mixture was stirred under 30 psi ofhydrogen for 45 minutes. The catalyst was filtered off and the residueflash chromatographed eluting with dichloromethane:methanol (30:1) toobtain the title compound (57 mg).

δ (¹ H, CDCl₃): 9.88 (s, 1H, CHO), 6.04 (dd, 1H, H-2, J=1.5 and 3.6 Hz),4.69 (dd, 1H, H-1', J=2.1 and 9.6 Hz), 4.46 (d, 1H, 8aCH₂, J=9.3 Hz),3.93-3.90 (m, 1H, H-3'), 3.81 (dq, 1H, H4', J_(d) =9.6 Hz, J_(q) =6.6Hz), 3.61 (t, 2H, 3'--O--CH₂, J=7.2 Hz), 3.75 (s, 3H, 4--O--CH₃), 3.29(d, 1H, 8aCH₂, J=9.3 Hz), 2.84 (dd, 1H, H4', J=3.3 and 9.6 Hz), 2.43 (t,1H, H-1, J=3.9 Hz); δ (¹³ C, CDCl₃): 204.5 (CHO), 173.6 (CO₂ H), 148.5(C-3), 130.4 (C-2), 97.7 (C-1'), 82.5 (C-4'), 74.9 (C3a), 73.1 (8aCH₂),70.9 (C-3'), 69.4 (C-4'), 65.4 (C8a), 64.9 (3'--O--CH₂), 59.0 (C-4),57.1 (4'--O--CH₃).

EXAMPLE 7

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,6-Dideoxy-4-O-3-O-propyl-β-D-alopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

Sodium hydride (24 mg) was added portionwise to a solution ofIntermediate 10 (142 mg) in anhydrous tetrahydrofuran (5 ml) undernitrogen atmosphere at 0° C. The resulting suspension was stirred for 15minutes and allyl bromide (173 μl) was added. After 2 hours of stirring1N ammonium chloride (10 ml) and ethyl acetate (20 ml) were added. Theaqueous phase was extracted with ethyl acetate (2×20 ml) and the organicphase was washed with brine, dried (magnesium sulphate) and evaporatedto dryness. The residue was dissolved in ethyl acetate (10 ml) and 10%palladium on charcoal (100 mg) was added. The mixture was stirred under30 psi of hydrogen for 45 minutes. The catalyst was filtered off and theresidue flash chromatographed eluting with dichloromethane:methanol(30:1) to obtain the title compound (90 mg).

δ (¹ H, CDCl₃): 9.87 (s, 1H, CHO), 6.04 (dd, 1H, H-2, J=1.5 and 3.6 Hz),4.68 (dd, 1H, H-1', J=2.1 and 9.6 Hz), 4.44 (d, 1H, 8aCH₂, J=9.6 Hz),3.93-3.85 (m, 1H, H-3'), 3.81 (dq, 1H, H-5', J_(d) =9.3 Hz and J_(q)=6.3 Hz), 3.49 (t, 2H, 3'--O--CH₂, J=6.6 Hz), 3.70 (s, 3H, 4'--O--CH₃),3.29 (d, 1H, 8aCH₂, J=9.6 Hz), 2.84 (dd, 1H, H4', J=2.7 and 9.3 Hz),2.43 (t, 1H, H-1, J=4.2 Hz); δ (¹³ C, CDCl₃): 204.5 (CHO), 173.6 (CO₂H), 148.5 (C-3), 130.4 (C-2), 97.8 (C-1'), 82.6 (C4'), 74.8 (C3a), 73.2(8a-CH₂), 71.8 (3'--O--CH₂), 71.0 (C-3'), 69.4 (C-5'), 65.0 (C-8a), 59.0(C-4), 57.0 (4--O--CH₃).

EXAMPLE 8

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,6-Dideoxy-4-O-methyl-3-O-methyloxymethyl-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 11 (180 mg) in ethyl acetate (20 ml) wasadded 10% palladium on charcoal (100 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 20 psi of hydrogen for 40 minutes atroom temperature. The catalyst was filtered off and the solventevaporated to dryness. The residue was purified by flash columnchromatography on silica gel eluting with methylene chloride:methanol(20:1) to give the title compound (100 mg).

δ (¹ H, CDCl₃): 9.83 (s, 1H, CHO), 6.04 (dd, 1H, H-2, J=1.2 and 3.3 Hz),4.72 (AB system, 2H, OCH₂ OCH₃, J_(AB) =6.9 Hz), 4.68 (dd, 1H, H-1',J=2.1 and 9 Hz), 4.33 and 3.36 (2d, 2H, 8a-CH₂, J=9.6 Hz), 4.21 (m, 1H,H-3'), 3.79 (m, 1H, H-5'), 3.39 (s, 3H, OCH₃), 3.38 (s, 3H, OCH₃), 2.84(dd, 1H H4', J=2.7 and 9.3 Hz), 2.49 (t, 1H, H-1); δ (¹³ C, CDCl₃):204.5 (CHO), 173.8 (COOH), 148.4 (C-3), 130.4 (C-2), 97.8 (C-1'), 95.9(OCH₂ OCH₃), 82.4 (C4'), 73.3 (8a-CH₂), 69.2 (C-3'), 68.8 (C-5'), 65.0(C-8a), 57.3 (OCH₂ OCH₃), 55.5 (C-7), 47.2 (C-1), 41.6 (C4), 35.8(C-2'), 18.0 (C6').

EXAMPLE 9

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,6-Dideoxy-3-O-methyl-4-O-propyl-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of the 3-O-methyl compound from Intermediate 10 (120 mg)in dry tetrahydrofuran (20 ml) at 0√ C. was added sodium hydride (9 mg).The suspension was stirred for 0.5 hours and allyl bromide (10equivalents) and a catalytic amount of tetrabutylammonium iodide wereadded. The mixture was stirred for 2 days at room temperature. Thereaction was quenched with 1N ammonium chloride and the mixtureextracted with ethyl acetate. The organic layer was concentrated to 15ml and 10% palladium on charcoal was added (100 mg) under nitrogen. Themixture was shaken in a Parr apparatus under 15 psi of hydrogen for 30minutes at room temperature. The catalyst was filtered off and thesolvent evaporated to dryness. The residue was purified by flash columnchromatography on silica gel eluting with methylene chloride:methanol(20:1) to give the title compound (60 mg).

δ (¹ H, CDCl₃): 9.86 (s, 1H, CHO), 6.04 (dd, 1H, H-2, J=0.9 and 3.0 Hz),4.65 (dd, 1H, H-1, J=2.1 and 9.9 Hz), 4.42 and 3.80 (2d, 2H, 8a-CH₂,J=9.6 Hz), 3.76 (m, 1H, H-3'), 3.60-3.50 (m, 1H, H5'), 3.45 (s, 3H,OCH3), 3.38 -3.27 (m, 2H, OCH₂ CH₂ CH₃), 2.93 (dd, 1H, H-4', J=2.8 and9.6 Hz), 2.44 (t, 1H, H-1); δ (¹³ C, CDCl₃): 204.5 (CHO), 173.2 (COOH),148.5 (C-3), 130.3 (C-2), 97.5 (C-1'), 81.2 (C-4'), 73.8 (C-3'), 69.4(C-5'), 64.9 (C-8a), 34.4 (C-2), 18.0 (C-6').

EXAMPLE 10

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-Azido-3,6-dideoxy-4-O-methylβ-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A stirred solution of Intermediate 16 (0.29 mmol) in water (4 ml) at 0°C. was treated with trifluoroacetic acid (6 ml). After 90 minutes, themixture was poured into ether:water (1:1; 50 ml). The organic phase wasevaporated and the residue purified by flash column chromatography usingdichloromethane:methanol (50:1) as eluent to give the title compound (98mg).

δ (¹ H, CDCl₃): 9.68 (s, 1H, CHO), 6.07 (dd, 1H, H-2, J-=1.2 and 3.3Hz), 4.52 (d, 1H, H1', J=1.2 Hz), 4.18 (t, 1H, H-2', J=4.2 Hz), 4.02 and3.65 (m,m, 1H, 1H, 8a-CH₂), 3.78 (m, 2H, H-3' and H-5'), 3.46 (s, 3H,OCH₃), 3.37 (dd, 1H, H4', J=3.3 and 8.7 Hz), 2.67 (sbr, 1H, OH), 2.34(m, 1H, CH(CH₃)₂); δ (¹³ C, CDCl₃): 204.9 (CHO), 130.5 (C-2), 97.8(C-1'), 79.8, 69.7, 69.4 and 59.2 (C-2', C-3', C-4' and C-5'), 74.4(8a-CH₂), 65.6 (C-8a).

EXAMPLE 11

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3,6-Dideoxy-4-O-methyl-3-methylthio-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A stirred solution of Intermediate 17 (0.2 mmol) in water (4 ml) at 0°C. was treated with trifluoroacetic acid (6 ml). After 90 minutes, themixture was poured into ether:water (1:1; 50 ml). The organic phase wasevaporated and the residue purified by flash column chromatography usingdichloromethane:methanol (50:1) as eluent to give the title compound (68mg).

δ (¹ H, CDCl₃): 9.73 (s, 1H, CHO), 6.08 (dd,1H, H-2, J=1.2 and 3.3 Hz),4.74 (d, 1H, H-1', J=1.5 Hz), 4.09 and 3.64 (d,d, 1H, 1H, 8a-CH₂, J=9.6Hz), 3.96 ((dd, 1H, H-2', J=1.2 and 4.2 Hz), 3.72 (m, 1H, H-5'), 3.45(dd, 1 H, H-4', J=4.2 and 8.1 Hz), 3.40 (s, 3H, OCH₃), 3.33 (t, 1H,H-3', J=4.2 Hz), 2.66 (t, 1H, H-1, J=3.9 Hz), 2.34 (m, 1H, CH(CH₃)₂); δ(¹³ C, CDCl₃): 204.5 (CHO), 176.6 (CO₂ H), 148.2 (C-3), 130.7 (C-2),97.8 (C-1'), 79.5, 70.9, 70.4 and 57.4 (C-2', C-3', C4' and C-5'), 74.0(8a-CH₂), 72.3 (C-3a), 65.6 (C-8a), 58.8 (C-4).

EXAMPLE 12

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-Azido-3,6-dideoxy-4-O-methyl-β-D-glucopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A stirred solution of Intermediate 18 (0.37 mmol) in water (4 ml) at 0°C. was treated with trifluoroacetic acid (6 ml). After 90 minutes, themixture was poured into ether:water (1:1; 50 ml). The organic phase wasevaporated and the residue purified by flash column chromatography usingdichloromethane:methanol (50:1) as eluent to give the title compound(119 mg).

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 6.04 (dd, 1H,H-2, J=1.2 and 3.3 Hz),4.07 (d, 1H, H-1', J=7.5 Hz), 3.99 and 3.66 (d,d, 1H, 1H, 8a-CH₂, J=9.6Hz), 3.55 (s, 3H, OCH₃), 3.30 (m, 3H, H-2', H-3' and H-5'), 2.71 (m, 3H,H4', H-1 and 2'--OH), 2.31 (m, 1H, CH(CH₃)₂); δ (¹³ C, CDCl₃): 204.9(CHO), 174.3 (CO₂ H), 148.6 (C-3), 130.3 (C-2), 102.7 (C-1') 84.2, 72.9,72.2 and 67.7 (C-2', C-3', C4' and C-5'), 75.2 (8a-CH₂).

EXAMPLE 13

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3,6-Dideoxy-4-O-methyl-3-methylthio-β-D-glucopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A stirred solution of Intermediate 19 (0.17 mmol) in water (4 ml) at 0°C. was treated with trifluoroacetic acid (6 ml). After 90 minutes, themixture was poured into ether:water (1:1; 50 ml). The organic phase wasevaporated and the residue purified by flash column chromatography usingdichloromethane:methanol (50:1) as eluent to give the title compound (69mg).

δ (¹ H, CDCl₃): 9.74 (s, 1H, CHO), 6.09 (dd, 1H, H-2, J=1.2 and 3.3 Hz),4.14 (m, 2H, H-1' and 8a-CHa), 3.62 (m, 4H, 8a-CHb and OCH₃), 3.33 (m,1H, H-5'), 3.24 (dd, 1H, H-2', J=7.5 and 10.8 Hz), 2.80 (dd, 1H, H4, J=9and 10.2 Hz), 2.71 (t, 1H, H-1, J=3.6 Hz), 2.48 (t, 1H, H-3', J=10.5Hz), 2.33 (m, 1H1, CH(CH₃)₂), 2.20 (s, 3H, SCH₃); δ (¹³ C, CDCl₃): 204.7(CHO), 175.8 (CO₂ H), 148.3 (C-3), 130.7 (C-2), 103.7 (C-1'), 83.2(OCH₃), 74.7 (C-3a), 74.3, 70.9, 60.8 and 55.3 (C-2', C-3', C4' andC-5'), 73.0 (8a-CH₂), 65.6 (C-8a), 59.0 (C-4).

EXAMPLE 14

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-Acetylamino-3,6-dideoxy-4-O-methyl-β-D-glucopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A solution of Intermediate 20 (0.26 mmol) and sodium methoxide (0.6mmol) in dry methanol (3 ml) was stirred for 90 minutes at roomtemperature. The solvent was evaporated and the residue purified byflash chromatography using dichloromethane:methanol (13:1) as eluent togive the title compound (75 mg).

δ (hu 1H, CDCl₃): 9.74 (s, 1H, CHO), 6.09 (m, 2H, H-2 and NH), 4.17 (d,1H, H-1', J=7.5 Hz), 4.13 (t, 1H, H-3', J=7.2 Hz), 3.87 (m, 1H, H-2'),3.60 (d, 1H, 8a-CHa, J=9.6 Hz), 3.40 (m, 5H, H-5', 8a-CHb and OCH₃),2.96 (t, 1H, H4', J=9.6 Hz), 2.68 (t, 1H, H-1, J=3.9 Hz), 2.32 (m, 1H,CH(CH₃)₂), 2.07 (s, 3H, CH₃ CO); δ (¹³ C, CDCl₃): 204.7 (CHO), 174.9(CO₂ H), 172.5 (CO₂ CH₃), 148.3 (C-3), 130.7 (C-2), 103.6 (C-1'), 82.0(OCH₃), 74.8 (8a-CH₂), 73.5, 72.0, 58.8 and 56.7 (C-2', C-3', C-4' andC-5'), 73.2 (C-3a), 65.0 (C-8a), 58.9 (C-4).

EXAMPLE 15

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-Azido-4-O-methyl-2,3,6-trideoxy-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1)-carboxylicacid

Trifluoroacetic acid (0.1 ml) was added to Intermediate 23 (120 mg) indichloromethane (10 ml) at 0° C. After being stirred for 40 minutes themixture was quenched with a 5% aqueous sodium hydrogen carbonatesolution to pH7. The organic phase was dried and concentrated to give anoil, which was purified by flash column chromatography eluting withhexane:ethyl acetate (10:1) and dichloromethane:methanol (20:1) affordedthe title compound (68 mg) as a white foam.

δ (¹ H, CDCl₃): 9.80 (s, 1H, CHO), 6.05 (dd, 1H, H-2, J=1.2 and 3.6 Hz),4.55 (dd, 1H, H-1', J=1.8 and 9.3 Hz), 3.41 and 4.22 (m and d, 3H, 8aCH₂and H-3'), 3.75 (dq, 1H, H-5', J=6.3 and 9 Hz), 3.44 (s, 3H, 4'--OMe),3.00 (dd, 1H, H4', J=3 and 9 Hz); δ (¹³ C, CDCl₃): 204.5 (CHO), 174 (CO₂H), 148 (C-3), 130.6 (C-2), 97.3 (C-1'), 82.6 (C4'), 69.3 (C-5'), 59.1(C-3'), 57.6 (4'O--CH₃), 35.2 (C-2'), 17.9 (C-6').

EXAMPLE 16

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-3-O-(2-tetrahydropyranl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 25 (0.300 g) in ethyl acetate (20 ml) wasadded 10% palladium on charcoal (26 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 20 psi of hydrogen for 2 hours at roomtemperature. The catalyst was filtered off and the solvent evaporated todryness. The residue was purified on a silica gel flash column elutingwith 5% dichloromethane in methanol to afford the title compound (0.027g) as a 3:2 mixture of diastereomers.

δ (1H, CDCl₃) only major diastereomer: 9.75 (s, 1H, CHO), 6.07 (m, 1H,H-1"), 4.74 (m, 1H, H-2), 4.55 (m, 1H, H-1'); δ (¹³ C, CDCl₃): 206.6(CHO), 175.9 (CO₂ H), 150.1 (C-3), 131.6 (C-2), 102.1, 100.5 (C-1"),98.6 (C-1'), 81.3, 80.6 (C-3', C-4'), 76.4, 76.1 (8aCH₂, C-6).

EXAMPLE 17

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,3,6-Trideoxy-2,3-difluoro-4-O-methyl-β-D-glucopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 26 (95 mg) in ethyl acetate (25 ml) wasadded 10% palladium on charcoal (120 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 20 psi of hydrogen for 2 hours. Thecatalyst was filtered off and a mixture of 2N hydrochloric acid (1 ml)and methanol (5 ml) was added to the filtrate. The mixture was stirredat room temperature for 6 hours and then neutralized with a 5% aqueoussodium hydrogen carbonate solution. The solvent was evaporated todryness and the residue was flash chromatographed on silica gel elutingwith dichloromethane:methanol (20:1) to give the title compound (42 mg)as a white foam.

δ (¹ H, CDCl₃): 9.69 (s, 1H, CHO), 5.87 (dd, 1H, H-2, J=1.2 and 3.6 Hz),4.58-4.70 and 4.41-4.52 (2m, 1H, H-2', J_(FH) =52.8 and 17.1 Hz, J_(HH)=8.4 Hz), 4.25 (d, 1H, H-1', J=9 Hz), 4.30-4.40 and 4.10-4.23 (2m, 1H,H-3', J_(FH) =52.5 and 14.1 Hz, J_(HH) =7.8 and 8.1 Hz), 3.69 and 4.01(2d, 2H, 8aCH₂, J=9.3 Hz), 3.55 (s, 3H, 4'--OCH₃), 3.21-3.31 (m, 1H,H-5'), 2.97-3.07 (dq, 1H, H-4', J_(FH) =13.2 Hz, J_(HH) =8.4 and 8.7Hz), 2.81 (t, 1H, H-1); δ (¹³ C, CDCl₃): 204.7 (CHO), 177.1 (CO₂), 147.9(C-3), 131.2 (C-2), 100.0 (dd, C-1', J_(CF) =11 and 22 Hz), 96.7 and97.3 (dd, C-2', J_(CF) =18 and 185 Hz), 91.5 and 89.1 (dd, C-3', J_(CF)=18 and 189 Hz), 83.0 (dd, C-4', J_(CF) =6 and 16 Hz), 75.4 (8aCH₂),72.1 (C-8a), 70.0 (d, C-5', J_(CF) =9 Hz), 65.8 (C-3a), 60.6 (4'OMe),58.4 (C-4).

EXAMPLE 18

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,3,6-Trideoxy-4-O-methyl-3-oxo-β-D-glucopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 27 (0.17 mmol) in ethyl acetate (20 ml)was added 10% palladium on charcoal (50 mg) under nitrogen. The mixturewas shaken in a Parr apparatus under 20 psi of hydrogen for 1 hour atroom temperature. The catalyst was filtered off and the solventevaporated to dryness. The residue was purified by flash chromatographyusing dichloromethane:methanol (20:1) as eluent to give the titlecompound (63 mg).

δ (¹ H, CDCl₃): 9.72 (s, 1H, CHO), 6.06 (dd, 1H, H-2, J=1.2 and 3.3 Hz),4.48 (dd, 1H, H-1', J=2.7 and 9 Hz), 4.05 and 3.62 (d,d, 1H, 1H, 8a-CH₂,J=9 Hz), 3.43 (m, 2H, H-4' and H-5'), 2.65 (m, 3H, CH₂ --2' and H-1),2.32 (m, 1H, CH(CH₃)₂), 1.42 (d, 3H, CH₃ --6', J=5.7 Hz); δ (¹³ C,CDCl₃): 204.5 (CHO), 204.0 (CO-3'), 176.6 (CO₂ H), 148.2 (C-3), 130.8(C-2), 100.3 (C-1'), 87.0 (C-4'), 74.6 (8a-CH₂), 71.4 (C-5'), 48,4(C-2').

EXAMPLE 19

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3,6-Dideoxy-4-O-methyl-3-oxo-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a suspension of 10% palladium on charcoal (100 mg) in ethyl acetate(10 ml) was added a solution of Intermediate 28 (141 mg) in ethylacetate (5 ml) and the mixture was hydrogenated at room temperatureunder 30 psi of hydrogen for 45 minutes. The catalyst was filtered offand the solvent evaporated to dryness. The residue was flashchromatographed on silica gel eluting with dichloromethane anddichloromethane:methanol (30:1) to obtain the title compound (83 mg).

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 6.11 (dd, 1H, H-2, J=1.5 and 3.6 Hz),4.16-4.11 (m, 2H, H-1, H-2'), 3.55 (s, 3H, 4'--O--CH₃), 3.57 (dd, 1H,H4', J=1.5 and 9.6 Hz), 3.40 (dq, 1 H, H4', J_(d) =9.6 Hz, J_(q) =6.0Hz), 2.82 (t, 1H, H-1, J=3.4 Hz); δ (¹³ C, CDCl₃): 205.0 (CHO), 204.6(C-3'), 175.6 (CO₂ H), 148.0 (C-3), 131.2 (C-2), 104.8 (C-1'), 86.1(C4'), 75.3 (8aCH₂), 72.3 (C3a), 71.7 (C-5'), 65.4 (C8a), 59.6 (CH₃ O).

EXAMPLE 20

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3,6-Dideoxy-4-O-methyl-3-propyloxyimino-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

Intermediate 31 (180 mg) was dissolved in ethyl acetate (15 ml) and 10%palladium on charcoal (100 mg) was added. The mixture was stirred under30 psi of hydrogen atmosphere for 3 hours. The catalyst was filtered offand the solvent evaporated to dryness. The residue was dissolved intetrahydrofuran (5 ml) and 1N hydrochloric acid (3 ml) was added. Themixture was stirred for 4 hours at room temperature, diluted with waterand extracted with ethyl acetate (3×25 ml). The organic phase was washedwith brine, dried (magnesium sulphate) and evaporated to dryness. Theresidue was flash chromatographed on silica gel eluting withdichloromethane and dichloromethane:methanol (30:1) to obtain the titlecompound (107 mg) as a mixture of Z:E isomers in a 3:1 ratio.

δ (¹ H, CDCl₃) peaks of the major component: 9.75 (s, 1H, CHO), 6.08(dd, 1H, H-2, J=1.5 and 3.6 Hz), 4.79 (d, 1H, H-1', J=5.1 Hz), 4.59 (dd,1H, H-2', J=0.6 and 5.1 Hz), 4.17-4.08 (m, 5H, OCH₂, H-4', H-5', 8aCH₂),3.55 (d, 1H, 8aCH₂, J=9.3 Hz), 3.33 (s, 3H, 4--O--CH₃); δ (¹³ C, CDCl₃)peaks of the major component: 204.6 (CHO), 175.9 (CO₂ H), 151.1 (C=N),148.0 (C-3), 130.9 (C-2), 101.9 (C-1), 81.6 (C-4'), 76.3, 76.0 (C3a),74.9 (C-5'), 74.2 (8aCH₂), 65.8 (NOCH₂), 65.7 (C8a), 59.0 (CH₃ O).

EXAMPLE 21

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-Benzoyl-6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a-(1H)-carboxylicacid

To a solution of Intermediate 32 (250 mg) in methanol (30 ml), was added10% palladium on charcoal (120 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 10 psi of hydrogen for 1.5 hours atroom temperature. The catalyst was filtered off and the solventevaporated to dryness. The residue was purified by flash chromatographyeluting with hexane:ethyl acetate (4:1) and dichloromethane:methanol(15:1) to give the title compound (140 mg) as a white foam.

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 8.04 and 7.61-7.43 (2m, 2H and 3H,Ph), 6.05 (dd, 1H, H-2, J=3.3 and 1.2 Hz), 5.72 (dd, 1H, H-3', J=3 and0.9 Hz), 4.71 (s, 1H, H-1'), 3.72 and 4.06 (2d, 2H, 8aCH₂, J=9 Hz),3.87-3.94 (m, 2H, H-2' and H-5'), 3.38 (s, 3H, 4'--OMe), 3.41 (m, 1H,H-4'), 2.73 (t, 1H, H-1, J=3.9 Hz.); δ (¹³ C, CDCl₃): 204.8 (CHO), 175.8(CO₂ H), 165.3 (CO₂ of 3'OBz), 148.2 (C-3), 133.3, 129.6 and 128.4 (CHof Ph), 130.8 (C-2), 129.8 (quartemary of PH), 98.3 (C-1'), 78.3 (C4'),74.3 (8aCH₂), 72.2 (C-3a), 69.8 (C-3'), 69.2 (C-5'), 68.0 (C-2'), 65.6(C-8a), 58.9 (4'--OMe), 57.7 (C4), 46.1 (C-1).

EXAMPLE 22

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-Benzyloxycarbonyl-6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a,-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A stirred solution of Intermediate 33 (0.4 mmol) in water (4 ml) at 0°C. was treated with trifluoroacetic acid (6 ml). After 30 minutes, thesolution was poured into water:ether (1:1;50 ml). The organic phase wasevaporated and the residue purified by flash column chromatography usinghexane:ethyl acetate (1:2) as eluent to give the title compound (143mg).

δ (¹ H, CDCl₃): 9.69 (s, 1H, CHO), 7.37 (m, 5H, Ph), 6.04 (dd, 1H, H-2,J=1.2 and 3.3 Hz), 5.32 (t, 1H, H-3', J=3.3 Hz), 5.21 and 5.14 (ABsistem, 1H, 1H, CH₂ Ph, J=11.7 Hz), 4.62 (d, 1H, H-1', J=0.9 Hz), 4.03and 3.65 (d,d, 1H, 1H, 8a-CH₂, J=9 Hz), 3.87 (d, 1H, H-2', J=4.5 Hz),3.74 (m, 1H, H-5'), 3.28 (dd, 1H, H4', J=3 and 9 Hz), 2.67 (sbr, 1H,H-1), 2.33 (m, 1H, CH(CH₃)₂); δ (¹³ C, CDCl₃): 204.9 (CHO), 154.4 (CO3),134.9 (Cipso), 130.6 (C-2), 128.64, 128.60 and 128.41 (Ar), 98.0 (C-1'),78.2 (C-4'), 74.3 (8a-CH₂), 71.5 (C-5'), 70.0 (OCH₂ Ph), 69.3 (C-2'),69.0 (C-3').

EXAMPLE 23

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-3-O-butoxyacetyl-4-O-methyl-β-D-altropyranosyl-oxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 35 (300 mg) in ethanol (50 ml) was added10% palladium on charcoal (220 mg) under nitrogen. The mixture wasshaken in Parr apparatus under 15 psi of hydrogen for 1 hour at roomtemperature. The catalyst was filtered off and the solvent evaporated todryness. The residue was purified by flash column chromatography elutingwith hexane:ethyl acetate (1:1) and dichloromethane:methanol (10:1) toyield the title compound (160 mg).

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 6.07 (d, 1H, H-2, J=3.3 Hz), 5.55(dd, 1H, H-3', J=4.2 and 3.3 Hz) 4.57 (d, 1H, H-1', J=0.6 Hz), 4.12 (ABsystem, 2H, OCH₂ CO₂, J=12 Hz), 3.68 and 4.01 (2d, 2H, 8aCH₂, J=9.3 Hz),3.80 (d, 1H, H-2', J=3.9 Hz), 3.68 (m, 1H, H-5'), 3.53 (t, 2H, CH₂ O,J=6.6 Hz), 3.35 (s, 3H, 4'--OCH₃), 3.24 (dd, 1H, H-4', J=9 and 3 Hz),2.70 (m, 1H, H-1); δ (¹³ C, CDCl₃): 204.6 (CHO), 176.4 (CO₂ H), 169.6(3'--CO₂), 148.2 (C-3), 130.7 (C-2), 98.1 (C-1'), 78.2 (C-4'), 74.2(8aCH₂), 72.1 (C-3a), 71.7 (OCH₂ CO₂), 69.5 (C-5'), 69.0 (C-3'), 68.0(CH₂ CH₂ O), 67.9 (C-2'), 65.6 (C-8a); 58.8 (C-4), 57.7 (4'--OCH₃), 46.1(C-1).

EXAMPLE 24

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ,)]8a-[(6-Deoxy-4-O-methyl-3'-O-octanoyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 36 (400 mg) in methanol (75 ml) was added10% palladium on charcoal (275 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 15 psi of hydrogen for 1.5 hours atroom temperature. The catalyst was filtered and the solvent evaporatedto dryness. The residue was purified by flash chromatography on silicagel eluting with dichloromethane:methanol (20:1). The appropriatefractions were combined and the solvent removed to give the titlecompound (200 mg).

δ (¹ H, CDCl₃): 9.68 (s, 1H, CHO), 6.06 (dd, 1H, H-2, J=1.2 and 3.3 Hz),5.48 (dd, 1H, H-3', J=3 and 4.5 Hz), 4.57 (d, 1H, H-1', J=1.5 Hz), 4.02(d, 1H, H-8aCH₂, J=9.3 Hz), 3.72 (m, 3H, H-5', H-2' and H-8aCH₂), 3.28(s, 3H, OCH₃), 3.26 (dd, 1H, H-4', J=3 and 9 Hz), 2.68 (t, 1H, H-1,J=3.3 Hz), 2.36 (m, 3H, H-14 and CH₂ CO), 1.36-1.22 (m, 13H, 5CH₂ and6'CH₃); δ (¹³ C, CDCl₃): 205.0 (CHO), 177.1 (COOH), 172.6 (COOR), 148.5(C-3), 130.5 (C-2), 98.4 (C-1'), 78.2 (C-4'), 74.5 (C8aCH₂), 69.6(C-2'), 69.2 (C-3'), 67.2 (C-5'), 65.6 (CH₂ CO), 57.6 (OCH₃), 34.2,28.8, 24.9, 22.5 (5CH₂), 18.1 (C-6'), 14.0 (CH₃).

EXAMPLE 25

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-3-O-(2-methylhexanoyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A solution of α-methylhexanoic acid (0.133 ml) and triethylamine (0.313ml) was added to a solution of Intermediate 24 (0.371 g) and2-chloro-1-methyl pyridinium iodide (0.267 g). The mixture was refluxedfor 2 hours. After cooling, the solvent was evaporated and the residuewas chromatographed on a silica gel column eluting with hexane:ethylacetate (5:1) to afford the 3'-O-acyl derivative (0.43 g), which wasdissolved in ethyl acetate (20 ml) and hydrogenated in a Parr apparatususing 10% palladium on charcoal (100 mg). After filtering off thepalladium catalyst the solvent was removed and the residue waschromatographed on a silica gel flash column eluting with 5%dichloromethane:methanol to give the title compound (0.235 g) as a 1:1mixture of diastereoisomers.

δ (¹ H, CDCl₃): 9.70 (s, 1H, CHO), 6.05 (m, 1H, H-2), 5.47 (m, 1H,H-3'), 4.56 (brs, 1H, H-1'), 4.01 (brd, 1H, 8aCH₂), 3.73 (m, 3H, H-2',5' and 8aCH₂), 3.32 (1, 3H, OMe), 3.26 (m, 1H, H4'), 2.70 (m, 1H, H-1),2.05 (sext., 1H, CHCO₂), δ (¹³ C, CDCl₃): 204.7 (CHO), 176.9 and 175.6(CO₂ H and CO₂ C), 148.4 (C-3), 130.5 (C-2), 98.4 (C-1'), 78.1 (C4),74.5 (8aCH₂), 72.4 (C-3a), 69.7, 69.3 and 66.9 (C-2', C-3' and C-5'),65.6 (C-8a), 58.8 (C4).

EXAMPLE 26

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-3-O-methoxyacetyl-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 37 (200 mg) in ethanol (30 ml) was added10% palladium on charcoal (170 mg) under nitrogen. The mixture wasshaken in Parr apparatus under 15 psi of hydrogen for 1 hour at roomtemperature. The catalyst was filtered off and the solvent evaporated todryness. The residue was purified by flash column chromatography elutingwith hexane:ethyl acetate (3:1) and dichloromethane:methanol (15:1) toyield the title compound (120 mg).

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 6.07 (dd, 1H, H-2, J=3.6 and 1.2 Hz),5.56 (dd, 1H, H-3', J=4.2 and 3.3 Hz), 4.57 (d, 1H, H-1', J=1.5 Hz),4.08 (AB system, 2H, OCH₂ CO₂, J=12 Hz), 3.68 and 4.02 (2d, 2H, 8aCH₂,J=9 Hz), 3.80 (dd, 1H, H-2', J=1.2 and 4.5 Hz), 3.71 (m, 1H, H-5'), 3.35(s, 3H, 4'--OCH₃), 3.45 (s, 3H, CH₃ CH₂ CO₂), 3.24 (dd, 1H, H4', J=9 and3 Hz), 2.57 (t, 1H, H-1, J=3.9 Hz); δ (¹³ C, CDCl₃): 204.7 (CHO), 175.9(CO₂ H), 169.2 (3'--CO₂), 148.2 (C-3), 130.7 (C-2), 98.0 (C-1'), 78.2(C4'), 74.1 (8aCH₂), 72.1 (C-3a), 69.5 (OCH₂ CO,), 69.4 (C-5'), 68.9(C-3'), 67.9 (C-2'), 65.6 (C-8a), 59.3 (CH₂ OCH₃), 58.9 (C-4), 57.7(4'--OCH₃), 46.0 (C-1).

EXAMPLE 27

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-3-O-((E)-2-methyl-2-hexenoyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

Zinc dust (300 mg) followed by 1M aqueous potassium dihydrogen phosphate(1 ml) were added to a rapidly stirred solution of Intermediate 39 (131mg) in tetrahydrofuran (5 ml) at room temperature. The resultant slurrywas stirred for, 3 days at room temperature and the solids were removedby filtration and washed with a (5:1) mixture of tetrahydrofuran:water(20 ml). The filtrate and washings were combined, the solvent wasremoved in vacuo and the residue evaporated twice from toluene. Theresulting colourless gum was purified by preparative tic (Merck 5717)eluting with dichloromethane:methanol (10:1) and washing the product offthe silica gel with ethyl acetate:methanol (8:1). Removal of the solventgave the title compound (51 mg) as a white foam.

δ (¹ H, CDCl₃): 9.72 (s, 1H, CHO), 6.8 (m, 1H, RO₂ CC(CH₃)=CH--R'), 6.06(m, 1H, H-2), 5.54 (m, 1H, H-3'), 4.60 (d, 1H, H-1', J=1.2 Hz), 4.09,3.67 (2d, 2H, 8aCH₂, J_(AB) =9.6 Hz), 3.84 (m, 1H, H-2'), 3.77 (m, 1H,H-5'), 3.35 (s, 3H, --OCH₃), 3.3 (m, 1H, H4'), 2.68 (m, 1H, H-1), 2.34(m, 1H, CHMe₂).

EXAMPLE 28

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-(4-Chlorobutyryl)-6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 40 (250 mg) in methanol (10 ml) was added10% palladium on charcoal (165 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 18 psi of hydrogen for 1 hour at roomtemperature. The catalyst was filtered and the solvent evaporated todryness. The residue was purified by flash chromatography on silica gel,eluting with dichloromethane:methanol (20:1). The appropriate fractionswere combined and the solvent removed to give the title compound (138mg) as a white foam.

δ (¹ H, CDCl₃): 9.69 (s, 1H, CHO), 6.07 (dd, 1H, H-2, J=1.2 and 3.3 Hz),5.49 (dd, 1H, H-3', J=3.6 and 4.5 Hz), 4.57 (d, 1H, H-1', J=1.5 Hz),4.00 (d, 1H, H-8aCH₂, J=9.3 Hz), 3.77 (dd, 1H, H-2', J=1.2 and 4.5 Hz),3.71 (m, 1H, H-5'), 3.69 (d, 1H, H-8aCH₂, J=9.3 Hz), 3.6 (t, 2H, CH₂ O,J=6.3 Hz), 3.33 (s, 3H, OCH₃), 3.25 (dd, 1H, H4', J=3 and 9 Hz), 2.69(m, 1H, H-1), 2.56 (t, 2H, CH₂ Cl, J=7.2 Hz); δ (¹³ C, CDCl₃): 204.4(CHO), 177.1 (CO₂ H), 171.5 (COO), 148.3 (C-3), 130.0 (C-2), 98.2(C-1'), 78.2 (C4'), 74.5 (C8aCH₂), 72.2 (C-3a), 69.5 (C-2'), 69.1(C-3'), 67.6 (C-5'), 65.6 (CH₂ CO), 58.6 (C-4), 57.7 (OCH₃), 46.0 (C-1),43.6 (CH₂ Cl), 31.9 (COCH₂ CH2CH₂ Cl), 18.1 (C-6')).

EXAMPLE 29

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-3-O-(2-methylpropanoyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 41 (0.270 g) in ethyl acetate (25 ml) wasadded 10% palladium on charcoal (25 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 25 psi of hydrogen for 2 hours at roomtemperature. The catalyst was filtered off and the solvent evaporated todryness. The residue was purified on a silica gel flash column elutingwith hexane:ethyl acetate (3:1) to afford the title compound (0.119 g).

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 6.06 (dd, 1H, H-2, J=0.9 and 3.3 Hz),5.46 (dd, 1H, H-3', J=3.0 and 4.5 Hz), 4.56 (d, 1H, H-1', J=1.5 Hz),4.00 (brd, 1H, 8aCH₂), 3.74 (m, 3H, H-2', 5' and 8aCH₂), 3.32 (s, 3H,OMe), 3.26 (dd, 1H, H-4', J=3.0 and 9.0 Hz), 2.70 (m, 1H, H-1), 2.61(sept, 1H, (CH₃)₂ CHCO₂); δ (¹³ C, CDCl₃): 204.6 (CHO), 177.0 and 175.9(CO₂ H and CO₂ --C), 148.4 (C-3), 130.5 (C-2), 98.4 (C-1'), 78.2 (C-4'),74.5 (8aCH₂), 72.4 (C-3a), 69.6, 69.2 and 67.1 (C-2', C-3' and C-5'),58.7 (C-4).

EXAMPLE 30

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-3-O-propionyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A solution of Intermediate 42 (274 mg) in ethyl acetate (20 ml) washydrogenated in a Parr shaker at room temperature for 1.5 hours over 10%palladium on charcoal (150 mg). The catalyst was removed by filtrationand the filtrate evaporated to a colourless gum. This was purified bypreparative tic (Merck 5717) eluting with dichloromethane:methanol(20:1). The product was washed off the silica gel with ethylacetate:methanol (5:1) and the solvent removed in vacuo to give thetitle compound (123 mg) as a white foam.

δ (¹ H, CDCl₃): 9.72 (s, 1H, CHO), 6.07 (dd, 1H, H-2, J=1.2 and 3.6 Hz),5.48 (dd, 1H, H-3', J=3 and 4.5 Hz), 4.58 (d, 1H, H-1', J=1.2 Hz), 4.01(d, 1H, A part of 8aCH₂, J_(AB) =9.3 Hz), 3.81-3.66 (m, 3H, H-2', H-5'and B part of 8aCH₂ (d)), 3.35 (s, 3H, OCH₃), 3.26 (dd, 1H, H-4', J=3and 9 Hz), 2.7 (bt, 1H, H-1, J=3.6 Hz), 2.46-2.26 (m, 3H, RO₂ CCH₂ Meand CHMe₂), 1.16 (t, 3H, RO₂ CCH₂ CH₃, J=7.5 Hz); δ (¹³ C, CDCl₃): 204.6(CHO), 176.4 (CO₂ H), 173.3 (CO₂ R), 148.2 (C-3), 130.7 (C-2), 98.3(C-1'), 78.3, 69.6, 69.2, 67.4 (C-2', C-3', C-4', C-5'), 74.2 (8aCH₂),72.2 (C-3a), 65.6 (C-8a), 58.8 (C4), 57.7 (OCH₃), 46.1 (C-1).

EXAMPLE 31

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-3-O-(trans-4-methyl-1-cyclohexanecarbonyl)-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 43 (297 mg) in ethyl acetate (20 ml) wasadded 10% palladium on charcoal (150 mg) under nitrogen. The mixture wasshaker in a Parr apparatus for 0.5 hours at room temperature. Thecatalyst was filtered off and the filtrate evaporated to dryness. Theresidue was purified by preparative tic (silica gel, Merck 5717) elutingwith dichloromethane:methanol (20:1) and extracted from the silica gelusing ethyl acetate:methanol (5:1). The solvent was removed in vacuo togive the title compound (149 mg) as a white foam.

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 6.06 (dd, 1H, H-2, J=1.2 and 3.3 Hz),5.45 (dd, 1H, H-3', J=3.3 and 4.5 Hz), 4.56 (d, 1H, H-1', J=1.5 Hz),4.01 (d, 1H, A part of 8aCH₂, J_(AB) =9.3 Hz), 3.8-3.64 (m, 3H, H-2',H-5' and B part of 8aCH₂), 3.32 (s, 3H, --OCH₃), 3.25 (dd, 1H, H4',J=3.3 and 8.7 Hz), 2.69 (bt, 1H, H-1, J=3.3 Hz), 2.4-2.2 (m, 2H, CHMe₂and RO₂ C--CH(cyclohexane)), 0.88 (d, 3H, H₃ C-cyclohexane-CO₂ R, J=6.3Hz); δ (¹³ C, CDCl₃): 234.7 (CHO), 176.6 (CO₂ H), 175 (RO₂C-cyclohexane), 148.3 (C-3), 130.6 (C-2), 98.4 (C-1'), 78.2, 69.7, 69.2,67 (C-2', C-3', C-4', C-5'), 74.4 (8aCH₂), 72.3 (C-3a), 65.6 (C8a), 58.8(C-4), 57.5 (OCH₃), 46.2 (C-1), 43.1 (RO₂ C--CH(cyclohexane).

EXAMPLE 32

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-trans-Cinnamoyl-6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

Zinc dust (400 mg) followed by 1M aqueous potassium dihydrogen phosphate(0.4 ml) were added to a vigorously stirred solution of Intermediate 45(170 mg) in tetrahydrofuran (4 ml) at room temperature. The resultantslurry was stirred for 2 days at room temperature and the solids wereremoved by filtration and washed with ethyl acetate. The filtrate andwashings were combined, the solvent was removed under vacuum and theresidue material partitioned between ethyl acetate (50 ml) and 1Naqueous hydrochloric acid (50 ml). The organic layer was washed withwater and brine, then dried (Na₂ SO4), filtered and evaporated. Theresidue was filtered through Dowex 50X8-200 ion-exchange resin usingmethanol as the eluent. The filtrate was concentrated in vacuo and theresidue flash chromatographed on silica gel eluting withdichloromethane:methanol (97:3) to give the title compound (105 mg) as awhite foam.

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 7.72 (d, 1H, Ph--CH=C--CO₂ R, J=15.9Hz), 7.58-7.36 (m, 5H, Ph), 6.48 (d, 1H, Ph--C=CH--CO₂ R), 6.08 (dd, 1H,H-2, J=1.5 and 3.6 Hz), 5.60 (dd, 1H, H-3', J=3 and 4.5 Hz), 4.66 (d,1H, H-1', J=1.2 Hz), 4.08, 3.7 (2d, 2H, 8aCH₂, J_(AB) =9 Hz), 3.92-3.78(m, 2H, H-2', H-5'), 3.39 (s, 3H, --OCH₃), 3.35 (dd, 1H, H-4', J=3 and8.7 Hz), 2.7 (t, 1H, H-1), 2.4-2.26 (m, 1H, CHMe₂); δ (¹³ C, CDCl₃):204.8 (CHO), 176 (--CO₂ H), 165.8 (RO₂ C--C=C), 148.2 (C-3), 145.8(Ph--C=C), 130.7 (C-2), 117.3 (RO₂ C--C=C), 98.3 (C-1'), 78.3, 69.7,69.2, 67.8 (C-2', C-3', C-4', C-5'), 74.2 (8aCH₂), 72 (3a), 65.6 (8a),58.9 (C-4), 57.7 (OCH₃), 46.1 (C-1).

EXAMPLE 33

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-3-O-methacryloyl-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

Zinc dust (300 mg) followed by 1M aqueous potassium dihydrogen phosphate(0.3 ml) were added to a rapidly stirred solution of Intermediate 47 (95mg) in tetrahydrofuran (4 ml) at room temperature. The resultant slurrywas stirred for 2 days at room temperature and the solids were removedby filtration and washed with ethyl acetate. The filtrate and washingswere combined, the solvent was removed under vacuum and the residue waspartitioned between ethyl acetate (50 ml) and 1N aqueous hydrochloricacid (50 ml). The organic layer was washed successively with water andbrine, then dried over sodium sulphate, filtered and evaporated. Theresidue was filtered through Dowex 50X8-200 ion-exchange resin usingmethanol as the eluent. The filtrate was concentrated by evaporation andthe residue was flash chromatographed on silica gel eluting withdichloromethane:methanol (96:4). Removal of the solvent gave the titlecompound (60 mg).

δ (¹ H, CDCl₃): 9.72 (s, 1H, CHO), 6.14 (bs, 1H, Ha--C=C), 6.07 (m, 1H,H-2), 5.62 (m, 1H, Hb--C=C), 5.54 (m, 1H, H-3'), 4.60 (d, 1H, H-1',J=0.9 Hz), 4.03,3.69 (2d, 2H, 8aCH₂, J_(AB) =9.3 Hz), 3.84 (d, 1H, H-2',J=4.5 Hz), 3.76 (m, 1H, H-5'), 3.35 (s, 3H, --OCH₃), 3.31 (dd, 1H, H-4',J=3 and 9 Hz), 2.69 (m, 1H, H-1), 2.34 (m, 1H, CHMe₂), 1.97 (s, 3H, H₃C--C=C); δ (¹³ C, CDCl₃): 204.8 (CHO), 176 (--CO₂ H), 166 (R--CO₂ R'),148.4 (C-3), 135.9 (--C=CH₂), 130.7 (C-2), 126.3 (--C=CH₂), 98.4 (C-1'),78.3, 69.8, 69.2, 67.8 (C-2', C-3', C-4', C-5'), 74.4 (8aCH₂), 72 (C3a),65.7 (C-8a), 58.9 (C4), 57.7 (OCH₃), 46.2 (C-1).

EXAMPLE 34

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα,8aβ)]8a-[(6-Deoxy-4-O-methyl-3'-O-octyloxycarbonyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 48 (320 mg) in methanol (40 ml) was added10% palladium on charcoal (175 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 15 psi of hydrogen for 1.5 hours atroom temperature. The catalyst was filtered and the solvent evaporatedto dryness. The residue was purified by flash chromatography on silicagel, eluting with dichloromethane:methanol (20:1). The appropriatefractions were combined and the solvent removed to give the titlecompound (168 mg) as a white foam.

δ (¹ H, CDCl₃): 9.66 (s, 1H, CHO), 6.04 (dd, 1H, H-2, J=0.9 and 3.3 Hz),5.28 (dd, 1H, H-3', J=3 and 3.9 Hz), 4.63 (d, 1H, H-1', J=0.9 Hz) 4.13(t, 2H, CH₂ OCO, J=6.6 Hz), 4.02 and 3.66 (2d, 2H, 8aCH₂, J=9.6 Hz),3.86 (dd, 1H, H-2', J=1.2 and 4.2 Hz), 3.74 (m, 1H, H-5'), 3.37 (s, 3H,OCH₃), 3.27 (dd, 1H, H-4, J=3 and 9 Hz), 1.4-1.1 (m, 6CH₂ and 6'CH₃); δ(¹³ C, CDCl₃): 205.1 (CHO), 177.3 (COOH), 154.5 (OCOO), 148.5 (C-3),130.5 (C-2), 98.2 (C-1'), 78.2 (C-4'), 74.5 (C8aCH₂), 72.6 (C-3a), 71.1(C-2'), 69.3 (C-3'), 69.1 (C-5'), 68.6 (CH₂ --OCOO), 57.9 (OCH₃), 31.6,29.1, 29.0, 28.5, 25.5, 22.5 (6CH₂ --), 17.9 (C-6'), 14.0 (CH₃).

EXAMPLE 35

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-4-O-methyl-3-O-octanoylaminocarbonyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 49 (200 mg) in ethyl acetate (20 ml) wasadded 10% palladium on charcoal (100 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 15 psi of hydrogen for 30 minutes atroom temperature. The catalyst was filtered off and the solventevaporated to dryness. The residue was chromatographed on a silica gelflash column eluting with methylene chloride and methylenechloride:methanol (15:1) to give the title compound (135 mg) as a whitefoam.

δ (¹ H, CDCl₃): 9.72 (s, 1H, CHO), 6.07 (dd, 1H, H2, J=1.2 and 2.4 Hz),5.32 (m, 1H, H-3'), 4.80 (t, 1H, CH₂ --NH--CO₂), 4.57 (d, 1H, H-1',J=1.2Hz), 4.02 and 3.66 (2d, 2H, 8a-CH2, J=9.6 Hz), 3.74 (m, 1H, H-5'),3.38 (s, 3H, 4'OCH₃), 3.28 (dd, 1H, H-4', J=2.8 and 8.7 Hz), 3.80 (m,2H, CH₂ NHCO₂), 2.70 (t, 1H, H-1); δ (¹³ C, CDCl₃): 204.7 (CHO), 175.9(COOH), 155.5 (OCONH, 148.2 (C-3), 130.7 (C-2), 93.7 (C-1), 78.5 (C-4'),72.2 (8a-CH₂), 69.6 (C-3'), 69.2 (C-5'), 65.6 (C-8a), 58.8 (C-2), 57.7(C-7'), 18.2 (C-6').

EXAMPLE 36

[1R-(1α, 3aβ, 4β, 4aβ7β, 7aα, 8aβ)]8a-[(6-Deoxy-3-O-benzylaminocarbonyl-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 50 (100 mg) in ethyl acetate (15 ml) wasadded 10% palladium on charcoal (50 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 15 psi of hydrogen for 10 minutes. Thecatalyst was filtered off and the solvent evaporated to dryness. Theresidue was chromatographed on a silica gel flash column using methylenechloride and methylene chloride:methanol (20:1) as eluents to give thetitle compound (56 mg) as a white foam.

δ (¹ H, CDCl₃): 9.70 (s, 1H, CHO), 7.28 (m, 5H, 3'--Ph), 6.06 (d, 1H,H-2, J=3.3 Hz), 5.37 (m, 1H, H-3'), 5.17 (t, 1H, Ph--NH--CO₂), 4.55 (s,1H, H-1'), 4.38 (m, 2H, Ph--CH₂ --NH), 3.99 and 3.66 (2d, 2H, 8a-CH₂,J_(AB) =9.3 Hz), 3.87 (d, 1H, H-2', J=4.5 Hz), 3.39 (s, 3H, 4'--OCH₃),3.28 (dd, 1H, H-4', J=2.7 and 8.4 Hz), 2.71 (t, 1H, H-1).

EXAMPLE 37

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(6-Deoxy-3-O-dimethylaminocarbonyl-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 51 (0.165 g) in ethyl acetate (20 ml) wasadded 10% palladium on charcoal (20 mg) under nitrogen. The mixture wasshaken in a Parr apparatus under 20 psi of hydrogen for 2 hours at roomtemperature. The catalyst was filtered off and the solvent evaporated todryness. The residue was purified on a silica gel flash column elutingwith 5% dichloromethane in methanol to give the title compound (0.060g).

δ (¹ H, CDCl₃): 9.71 (s, 1H, CHO), 6.07 (dd, 1H, H-2, 1.2 and 3.3 Hz),5.36 (dd, 1H, H-3', J=3 and 4.5 Hz), 4.57 (d, 1H, H-1', J=1.2 Hz), 4.02(m, 1H, 8aCH₂), 3.85 (m, 1H, H-2'), 3.75 (m, 1H, H-5'), 3.66 (m, 1H,8aCH₂), 3.37 (s, 3H, MeO), 3.28 (dd, 1H, H-4', J=3.3+9.0 Hz), 2.94 (s,6H, Me₂ N), 2.70 (m, 1H, H-1); δ (¹³ C, CDCl₃): 204.9 (CHO), 176.2 (CO₂H), 155.5 (CON), 148.4 (C-3), 130.7 (C-2), 98.6 (C-1'), 78.5 (C-4'),74.3 (8aCH₂), 72.4 (C-3a), 69.9, 69.4, 68.7 (C-2', C-3', and C-5'), 65.7(C-8a), 58.9 (C-4).

EXAMPLE 38

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,6-Dideoxy-4-O-methyl-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 33 (200 mg) in dry tetrahydrofuran (10 ml)at 0° C. and under nitrogen atmosphere, sodium hydride (25 mg) andimidazole (2 mg) were added. The solution was stirred for 10 minutes andcarbon disulfide (0.12 ml) was added. After 20 minutes methyl iodide(0.25 ml) was added and the stirring continued for 30 minutes. 1Nammonium chloride was added and the reaction mixture extracted withethyl acetate, the organic phase washed with brine, dried over magnesiumsulphate and the solvent evaporated to dryness. The residue was flashchromatographed eluting with hexane:ethyl acetate (4:1) to obtain 214 mgof xanthate. 200 mg of this compound were dissolved in dry toluene (8ml) under nitrogen atmosphere and heated at 110° C. A solution oftributyltin hydride (0.12 ml) in dry toluene (10 ml) was added dropwiseover 2.5 hours with stirring. Methanol was added and the reactionmixture evaporated to dryness. The major compound of the crude waspurified by flash chromatography on silica gel eluting with ethylacetate:hexane (15:85) to obtain a foam. This foam was dissolved inethyl acetate (10 ml). Palladium on charcoal was added (50 mg) and thesuspension stirred under hydrogen atmosphere (30 psi) for 2 hours. Thecatalyst was filtered off and the solvent removed. The crude was flashchromatographed on silica gel eluting with dichloromethane:methanol(96:4) to yield the title compound (41 mg) as a foam.

δ (¹ H, CDCl₃): 9.82 (s, 1H, CHO), 6.04 (dd, 1H, H-2, J=1.2 and 4.2Hz),4.71 (dd, 1H, H-1', J=2.4 and 9.9 Hz), 4.32 (d, 1H, 8aCH₂, J=9.0 Hz),4.24 (dd, 1H, H-3', J=3.0 and 6.6 Hz), 3.80-3.60 (m, 1H, H-5'), 3.40 (s,3H, CH₃ O), 3.55 (d, 1H, 8aCH₂, J=9.0 Hz), 2.85 (dd, 1H, H-4', J=3.0 and9.3 Hz), 2.49 (t, 1H, H-1, J=3.6 Hz); δ (¹³ C, CDCl₃): 204.6 (CHO),173.3 (CO₂ H), 148.4 (C-3), 130.5 (C-2), 97.6 (C-1'), 82.1 (C-4'), 73.5(8aCH₂), 68.5, 66.1, 63.8, 61.1, 57.3.

EXAMPLE 39

[1R-(1α, 3aβ, 4aβ, 7β, 7aα, 8aβ)]8a-[(2,3,6-Trideoxy-4-O-methyl-β-D-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 52(a) (180 mg) in ethyl acetate (40 ml)was added 10% palladium on charcoal (150 mg) under nitrogen. The mixturewas shaken in a Parr apparatus under 15 psi of hydrogen for 1 hour atroom temperature. The catalyst was filtered off and the solventevaporated to dryness. The residue was purified by flash columnchromatography eluting with hexane:ethyl acetate (3:1) anddichloromethane:methanol (10:1) to yield the title compound (82 mg).

δ (¹ H, CDCl₃): 9.84 (s, 1H, CHO), 6.04 (dd, 1H, H-2, J=1.2 and 3.3 Hz),4.33 (m, 2H, H-1' and H-8aCH₂), 3.34 (m, 4H, H-8aCH₂ and 4'--OCH₃), 3.28(dq, 1H, H-5', J=6.3 and 9Hz), 2.80 (m, 1 H, H-4'), 2.50 (t, 1H, H-1,J=3.9 Hz); δ (¹³ C, CDCl₃): 204.6 (CHO), 174.1 (CO₂ H), 148.4 (C-3),130.4 (C-2), 101.2 (C-1'), 79.8 (C-4'), 74.8 (C-5'), 73.1 (8aCH₂), 65.1(C-8a), 59.0 (C-4), 56.9 (4'--OCH₃), 47.1 (C-1), 41.0 and 41.5 (C-5 andC-6).

EXAMPLE 40

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3,6-dideoxy-4-O-methyl-β-D-altropyranoxyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

10% palladium on charcoal (100 mg) was added to a solution ofIntermediate 53 (100 mg) in ethyl acetate (30 ml) under nitrogen. Themixture was shaken in a Parr apparatus under 15 psi of hydrogen for 1hour at room temperature. The catalyst was filtered off and the solventevaporated to dryness. The residue was purified by flash columnchromatography eluting with hexane:ethyl acetate (3:1) anddichloromethane:methanol (15:1) to give the title compound (60 mg) as awhite foam.

δ (¹ H, CDCl₃): 9.74 (s, 1H, CHO), 6.06 (dd, 1H, H-2, J=1.2 and 3.3 Hz),4.35 (s, 1H, H-1'), 3.64 and 4.09 (2brd, 2H, BaCH₂, J=9Hz), 3.90 (m, 1H,H-2'), 3.35 (m, 4H, H-5' and 4'--OCH₃), 3.21 (m, 1H, H-4'), 2.67 (m, 1H,H-1), 2.41 (m, 1H, H-3'a); δ (¹³ C, CDCl₃): 204 (CHO), 175.9 (CO₂ H),148.4 (C-3), 130.6 (C-2), 100.6 (C-1'), 76.6 (C-4'), 74.4 (C-2'), 74.0(8aCH₂), 67.6 (C-5'), 65.6 (C-8a), 58.8 (C-4), 57.1 (4'--OCH₃).

EXAMPLE 41

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-(((2,3,6-Trideoxy-3-(tert-butoxycarbonyl)amino-b-D-allopyranosyl)oxy)methyl)-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of intermediate 22 (100 mg) in ethyl acetate (15 ml),di-tert-butyl dicarbonate (0.04 ml) and palladium (10%) on charcoal (30mg) were added under nitrogen. The mixture was shaken in a Parrapparatus (PH₂ =20 psi) for 2 hours at room temperature. The catalystwas filtered and the solvent evaporated to dryness. The residue waspurified by flash column chromatography on silica gel eluting withdichloromethane:methanol 20:1 to give pure title compound (20 mg).

δ (¹ H, CDCl₃): 13.20 (br s, 1H, COOH), 9.60 (s, 1H, CHO), 6.53 (br s,1H, NH), 6.04 (d, 1H, H-2, J=2.7 Hz), 4.82 (br s 1H, OH), 4.48 (d, 1H,H-1', J=6.3 Hz), 3.82 (m, 1H, H-4'), 3.70 (d, 1H, H-8a, J=9.0 Hz), 3.61(dq, 1H, H-5', J=8.1, 6.6 Hz), 3.48 (d, 1H, H-8a, J=9.0 Hz), 3.17 (m,1H, H-3'), 2.58 (t, 1H, H-1, J=3.3 Hz), 2.21 (m, 1H, CH(CH₃)₂); δ (¹³ C,CDCl₃): 204.8 (CHO), 176.0 (COO--), 175.7 (COOH), 148.1 (C-3), 130.7(C-2), 97.8 (C-1'), 73.7 (C-8a), 72.0 (C-5')+(C-4'), 46.47 (C-3').

EXAMPLE 42

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-(((2,3,6-Trideoxy-4-O-acetyl-3-acetylthio-b-D-allopyranosyl)oxy)methyl)-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A solution of Intermediate 54 (100 mg) in dichloromethane (10 ml) at 0°C. was treated with trifluoroacetic acid (0.1 ml). After 1 hour, themixture was washed with sodium bicarbonate saturated solution (3 ml).The organic layer was treated with brine (3 ml) and dried over magnesiumsulphate, filtered and concentrated. The residue was purified by flashcolumn chromatography on silica gel, using dichlromethane:methanol(20:1), to give pure title compound (18 mg).

δ (¹ H, CDCl₃)- 9.77 (s, 1H, CHO), 6.05 (dd, 1H, H-2, J=3.3 and 1.2 Hz),4.72 (dd, 1H, H-1', J=8.4 and 4.2 Hz), 4.48 (dd, 1H, H-3', J=6.3 and 4.5Hz), 4.30 (dd, 1H, H-4', J=8.7 and 4.5 Hz), 4.12 (d, 1H, H-8a, J=9.3Hz), 3.70 (dq, 1H, H-5', J=8.1 and 6.0 Hz), 3.49 (d, 1H, H-8a, J=9.3Hz), 2.60 (t, 1H, H-1, J=3.9 Hz), 2.35 (s, 3H, COCH₃), 2.33 (m, 1H,CH(CH₃)₂) 2.00 (s, 3H, COCH₃); δ (¹³ C, CDCl₃): 204.9 (CHO), 193.5(SCOCH₃), 174.7 (COOH), 169.8 (OCOCH₃), 148.4 (C-3), 130.6 (C-2), 98.7(C-1'), 73.7 (C-8a), 72.2 (C-4'), 70.5 (C-5'), 40.4 (C-3'), 30.7 (CH₃--COS), 20.8 (CH₃ --COO).

EXAMPLE 43

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-(3-Acetamido-2,3,6-trideoxy-4-O-methyl-β-D-allopyranosyloxy)methyl-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of intermediate 23 (0.75 mmol) in ethyl acetate (20 ml),palladium (10%) on charcoal was added (75 mg) under nitrogen. Themixture was shaken in a Parr apparatus (PH₂ =30 psi) for 4 hours at roomtemperature. The catalyst was filtered and the solution was treated withpyridine (3 ml) and acetic anhydride (0.2 ml) and stirred overnight atroom temperature. After this time, was washed with hydrochloric acid(2N), sodium bicarbonate and brine. After removal of the solvent, theresidue was purified by flash chromatography usingdichloromethane:methanol 20:1 to give the title compound (207 mg).

δ (¹ H, CDCl₃): 9.74 (s, 1H, CHO), 6.04 (dd, 1H, H-2, J=1.5 and 3.6 Hz),5.85 (d, 1H, NH, J=5.7 Hz), 4.50 (dd, 1H, H-1', J-2.8 and 7.8 Hz), 4.24(m, 1H, H-3'), 4.08 and 3.45 (d, d, 1H, 1H, 8a-CH₂, J=9.1 Hz), 3.87 (m,1H, H-5'), 3.32 (s, 3H, OMe), 3.04 (dd, 1H, H-4', J=4.2 and 7.5 Hz),2.60 (t, 1H, H-1, J=3.9 Hz), 2.04 (s, 3H, CH₃ CO); δ (¹³ C, CDCl₃):204.9 (CHO), 174.8 and 170.8 (CO₂ and CON), 148.3 (C-3), 130.7 (C-2),98.2 (C-1'), 79.6 (C-4'), 73.5 (8a-CH₂), 69.3 (C-5'), 65.3 (C-8a), 59.0(C-4).

EXAMPLE 44

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-benzoyl-2,6-dideoxy-4-O-methyl-β-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 55 (180 mg) in ethyl acetate (25 ml),palladium (10%) on charcoal (50 mg) was added. The mixture was shaken ina Parr apparatus (PH₂ =20 psi) for 1 hour at room temperature. Thecatalyst was filtered and the solvent evaporated to dryness. The residuewas purified by flash chromatography with mixtures of hexane:ethylacetate to give the title compound (60 mg) as a white foam.

δ (¹ H, CDCl₃): 9.8 (s, 1H, CHO), 8.03-7.5 (m, 5H, arom), 6.04 (dd, 1H,H-2, J=1.4 and 2.5 Hz), 5.7 (c, 1H, H-3'), 4.7 (dd, 1H, H-1'), 4.36,3.42 (dd, 2H, 8a-CH₂, J=10.8 Hz), 3.9 (m, 1H, H-5'), 3.39 (s, 3H, OCH₃),3.00 (dd, 1H, H-4'), 2.50 (t, 1H, H-1); δ (¹³ C, CDCl₃): 204.7 (CHO),173.64 (COOH), 165.53 (OCO--Ph), 148.5 (C-3), 130.5 (C-2), 133.2, 130.0,129.6, 128.4 (Carom), 97.78 8C-1'), 80.87 (C-4'), 73.5 (8a-CH₂), 69.9(C-5'), 66.17 (C-3'), 65.1 (C-8a), 57.5 (C4).

EXAMPLE 45

[1R-(1α, 3aβ, 4β, 4aβ, 7β, 7aα, 8aβ)]8a-[(3-O-acetyl-2,6-dideoxy-4-O-methyl-β-allopyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of Intermediate 56 (221 mg) in ethyl acetate (20 ml),palladium (10%) on charcoal (50 mg) was added. The mixture was shaken ina Parr apparatus (PH₂ =20 psi) for 1 hour at room temperature. Thecatalyst was filtered and the solvent evaporated to dryness. The residuewas purified by flash chromatography using hexane:ethyl acetate 3:7 togive the title compound (100 mg) as a colourless oil.

δ (1H, CDCl₃): 9.82 (s, 1H, CHO), 6.05 (dd, 1H, H-2, J=1.44 and 2.5 Hz),5.5 (c, 1H, H-3'), 4.63 (dd, 1H, H-1', J=2.5 and 9.36 Hz), 4.32, 3.39(d, d, 2H, 8a-CH,2, J=11 Hz), 3.72 (m, 1H, H-5'), 3.39 (s, 3H, OCH₃),2.88 (dd, 1H, H-4', J=3.6 Hz9, 2.5 (t, 1H, H-1), 2.1 (s, 2H, CH₃ CO); δ(13C, CDCl₃): 204.5 (CHO), 174.7 (COOH), 170.143 (COCH₃), 148.3 (C-3),130.56 (C-2), 97.67 (C1'), 80.78 (C4'), 73.8 (8aCH₂), 69.5 (C5'), 65.6(C3'), 65.19 (C8a), 57.56 (C4).

EXAMPLE 46

Characteristics of IMI 362184

IMI 362184 is a mutant of Sordaria araneosa (ATCC 36386, NRRL 3193)isolated following N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis ofascospores of this strain. The characteristics of IMI 362184 areessentially similar to those described in British Patent SpecificationNo. 1,162,027 for NRRL 3196, except that IMI 362184 produces4'-demethylsordarin as a major product under the same conditions usedfor sordarin production by NRRL 3196.

EXAMPLE 47

Characteristics of IMI 362947

IMI 362947 is a mutant of Sordaria araneosa (ATCC 36386, NRRL 3193)isolated following N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis ofascospores of this strain. The characteristics of IMI 362947 areessentially similar to those described in British Patent SpecificationNo. 1,162,027 for NRRL 3196, except that IMI 362947 does not produceascospores readily on agar. The strain also differs from NRRL 3196 inthat it produces sordaricin as a major product under the same conditionsused for sordarin production by NRRL 3196.

EXAMPLE 48

Characteristics of NCIMB 40675

NCIMB 40675 is an aerobic, Gram-positive, non-motile irregular rod thatproduces lemon yellow, translucent, round, entire, convex colonies witha diameter of between 0.5-1 mm when grown on tryptic soy agarsupplemented with 2% (w/v) yeast extract for 48 hours at 28° C. Theorganism grows well at temperatures up to 37° C., but not at 45° C.Metachromatic granules were not observed and the strain is catalasepositive, oxidase negative and does not metabolise glucosefermentatively. The strain can utilise the following sources of carbon:α-D-glucose, D-fructose, p-hydroxyphenyl-acetic acid, D-mannitol,methylpyruvate, lactamide, D-trehalose and sucrose. The organism canonly weakly utilise D-gluconic acid, pyruvic acid and salicin as solecarbon sources. Colony and microscopic morphology resembles that ofcoryneform bacteria. The genus Corynebacterium was excluded on thegrounds that the peptidoglycan of NCIMB 40675 contains ornithine ratherthan the meso-isomer of 2,6-diaminopimelic acid or diaminobutyric acid.Also, the organism contains a complex mixture of branch chain fattyacids atypical of Corynebacterium species, namely,12-methyltetradecanoic, 14-methylhexadecanoic and 14-methylpentadecanoicacids. The presence of α-branched-β-hydroxylated fatty acids was notdetermined. On the basis of these results NCIMB 40675 most closelyresembles one of the following actinobacterial genera: Aureobacterium,Curtobacterium or Cellulomonas.

To clarify the taxonomic position of NCIMB 40675, a 1100 base pairpartial sequence of the 16S rRNA gene was compared against 24 otherspecies representing a range of actinobacteria and related genera.Results from this analysis indicate a close relationship between NCIMB40675 and the genera Aureobacterium and Curtobacterium, but notCellulomonas or Corynebacterium. More precise identification for thestrain could be obtained by performing a phylogenetic analysis comparingvariable regions of the 16S rRNA gene for a range of Aureobactedum andCurtobacterium species in addition to further chemotaxonomic andphysiological tests.

Pharmacy Examples

    ______________________________________                                        1.      Conventional oral tablet                                              ______________________________________                                        Drug substance      100 mg                                                      Microcrystalline cellulose     160 mg                                         Crosscarmellose sodium           20 mg                                        Magnesium stearate                5 mg                                      ______________________________________                                    

The drug substance is blended with microcrystalline cellulose,crosscarmellose sodium and magnesium stearate, then compressed intotablets.

    ______________________________________                                        2.       Chewable oral tablet                                                 ______________________________________                                               Drug substance                                                                            100 mg                                                       Xylitol                    865 mg                                             Peppermint flavour            5 mg                                            Aspartame                    10 mg                                            Polyvinylpyrollidone         15 mg                                            Magnesium stearate            5 mg                                          ______________________________________                                    

The drug substance, xylitol, aspartame and polyvinylpyrollidone areblended together and granulated with water, then dried. This granule ismixed with the peppermint flavour and magnesium stearate, thencompressed into tablets.

    ______________________________________                                        3.      Aqueous Oral Solution                                                 ______________________________________                                        Drug substance        100 mg                                                    Hydroxypropylmethyl cellulose 150 mg                                          Sodium propylhydroxybenzoate   1 mg                                           Sodium methylhydroxybenzoate   2 mg                                           Orange flavour                10 mg                                           Sodium saccharin               5 mg                                           Sucrose                       800 mg                                          Suitable buffers                 qs                                           Purified water to              5 mls                                        ______________________________________                                    

Dissolve the drug substance and all the excipients in most of thepurified water and mix. Make to volume and mix. Suitable buffers may beadded to control the pH in the region of maximum stability.

    ______________________________________                                        4.      Non-Aqueous Oral Suspension                                           ______________________________________                                        Drug substance        100 mg                                                    Aspartame                    50 mg                                            Grapefruit flavour           25 mg                                            Mannitol                      800 mg                                          Colloidal silica               10 mg                                          Fractionated coconut oil        5 mls                                       ______________________________________                                    

Disperse the drug substance and mannitol in the bulk of the fractionatedcoconut oil by high shear mixing. Add the remaining ingredients and mix.Make to volume with fractionated coconut oil and mix.

    ______________________________________                                        5.       Ointment                                                             ______________________________________                                               Drug substance                                                                           200 mg                                                        White Soft Paraffin        9800 mg                                          ______________________________________                                    

Melt the white soft paraffin, add the drug and mix. Continue to mixuntil the ointment starts to congeal.

    ______________________________________                                        6.       Injection                                                            ______________________________________                                               Drug substance                                                                             40 mg                                                       Suitable buffers                  qs                                          Suitable antioxidants             qs                                          Suitable chelating agents         qs                                          Water for injections to         2 mls                                       ______________________________________                                    

Dissolve the drug substance in most of the water for injections.Suitable buffering agents may be added to control the pH to the regionof optimum stability. Suitable antioxidants and chelating agents may beadded to improve the stability of the injection. Make to mark with waterfor injections. Fill into ampoules or vials, then sterilise byautoclaving. Alternatively, sterilise by filtration and fillaseptically.

Antifungal Activity

Compounds of formula (I) have been tested for anti fungal activity in astandard in vitro screen and the minimum inhibiting concentration (MIC;μg/ml) determined for each compound against a variety of clinicallyrelevant pathogens. The results obtained with representative compoundsof the invention are given below.

The compounds of the invention are essentially non-toxic attherapeutically useful levels. For example the compounds of example 7,10 and 89 when administered at a dose of 50 mg/kg sc were active inprotecting male mice infected with C. albicans 4711E. At this dose noadverse effects were observed in the treated mice.

    __________________________________________________________________________                MICs μg/ml                                                       Example No.                                                                 ORGANISM    3    5   7   8   9   10  11  15  24  39  42                       __________________________________________________________________________    C. albicans 1208E                                                                         4.000                                                                              ≦0.001                                                                     0.015                                                                             0.250                                                                             8.0 1.0 1.0 ≦0.001                                                                     0.60                                                                              0.060                                                                             0.060                      C. albicans 2005E           0.250    ≦0.001  ≦0.001 0.030                                                             2.0       0.12                                                             0.250   ≦0.001                                                            ≦0.001                                                              0.004   ≦0.001      C. albicans 2402E           2.00     ≦0.001  0.008     0.250                                                              8.0       1.00                                                                1.00    ≦0.001                                                            0.030      0.060                                                            0.060                     C. albicans 4711            --      ≦0.001  0.004     0.120                                                               4.0       0.500                                                               1.00    ≦0.001                                                            --        --                                                               0.015                      C. tropicalis 2808E         1.00     0.004      0.003     2.00     16.0                                                               --    2.00                                                               0.004       0.060                                                               0.250   1.00                                                                 C. psuedotropicalis                                                          371E   0.250                                                                  ≦0.001  0.004                                                              0.120    2.0                                                                --   1.00                                                                   ≦0.001                                                                 0.004      0.008                                                              ≦0.001                                                                  C. glabrata 2375E                                                                    >125  1.000                                                               8.00      16.00                                                             >125   62                                                                   31.00   1.00                                                                  500        8.00                                                               31.00                      C. glabrata 2376E           >125  1.00       8.00      16.00    >125                                                             62     31.00   1.00                                                                 500                                                                     8.00    31.00                                                                  C. neoformans 2867E                                                                  31.00                                                                 31.00      125                                                                31.00    125                                                                  4.0    0.050   31.00                                                                0.25                                                                    62.00   >125             __________________________________________________________________________

We claim:
 1. A compound of the formula (I) ##STR14## andpharmaceutically acceptable salts and solvates or metabolically labilederivatives thereof,wherein R¹ represents hydrogen, halogen, hydroxyl orC₁₋₄ alkoxy; R² represents hydrogen, halogen, hydroxyl, C₁₋₁₀ alkoxy,C₁₋₁₀ alkylthio, C₁₋₆ alkoxyC₁₋₄ alkoxy, arylC₁₋₆ alkyloxy, arylC₃₋₆alkenyloxy, azido, NR⁵ COR⁵ where each R⁵ is independently hydrogen orC₁₋₆ alkyl, OR⁶ (where R⁶ is a cyclic ether containing 4 to 8 atomslinked to the oxygen atom via a ring carbon atom adjacent to the ringoxygen atom or a group ##STR15## where Y is oxygen, sulphur or NH, X iseither a bond, an oxygen atom or a moiety NR⁸ in which R⁸ is hydrogen orC₁₋₆ alkyl, and R⁷ is C₁₋₁₀ alkyl optionally containing one or twodouble bonds, aryl, arylC₁₋₄ alkyl, arylC₂₋₄ alkenyl, haloC₁₋₆ alkyl orC₁₋₆ alkoxyC₁₋₄ alkyl, and R³ represents hydrogen, or R² and R³ maytogether with the carbon atom to which they are attached represent C═Oor C═NOR⁹ where R⁹ is C₁₋₆ alkyl; and R⁴ represents hydroxyl, C₁₋₆alkoxy or ##STR16## (where R⁷ is as defined above); with the provisothat when R¹ represents a hydroxyl group in the axial configuration andR⁴ is methoxy then R² cannot represent a group in the axialconfiguration selected from hydroxyl and OCOCH═^(z) CH--CH═^(E) CHCH₃.2. A compound of the general formula (1a) ##STR17## and pharmaceuticallyacceptable salts and solvates (e.g. hydrates) thereof, wherein R¹ to R⁴are as defined in formula (I) above.
 3. A compound as claimed in claim 1wherein R¹ represents hydrogen or hydroxyl.
 4. A compound as claimed inclaim 1 wherein R² is in the axial configuration and R³ is hydrogen. 5.A compound as claimed in claim 1 wherein R² is C₁₋₆ alkoxy, C₁₋₆alkylthio, azido or OCOR⁷.
 6. A compound as claimed in claim 1 whereinR⁴ is C₁₋₄ alkoxy.
 7. A method of treatment of the human or non-humananimal body to combat fungal diseases which method comprisesadministering to said body an effective amount of a compound as claimedin claim
 1. 8. A pharmaceutical composition comprising a compound asclaimed in claim 1 in admixture with one or more physiologicallyacceptable carriers or excipients.
 9. A compound of formula (V)##STR18## or a protected derivative thereof.
 10. A process for thepreparation of a compound of formula (V) which comprises(a) cultivatinga microorganism capable of producing the compound of formula (V) andthereafter isolating the compound of formula (V) from the culture, (b)the biotransformation of sordarin.